Protease resistant modified interferon-beta polypeptides

ABSTRACT

Protease resistant modified interferon-beta polypeptides, pharmaceutical compositions and methods of treatment administering the compositions are provided. The modified interferon-beta polypeptides are orally available; hence pharmaceutical compositions formulated for oral administration also are provided.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/706,088, entitled, “RATIONAL EVOLUTION OF CYTOKINES FOR HIGHERSTABILITY, THE CYTOKINES AND ENCODING NUCLEIC ACID MOLECULES,” filedFeb. 13, 2007, now U.S. Pat. No. 7,998,469 which is a divisional of U.S.application Ser. No. 10/658,834, now U.S. Pat. No. 7,611,700, entitled,“RATIONAL EVOLUTION OF CYTOKINES FOR HIGHER STABILITY, THE CYTOKINES ANDENCODING NUCLEIC ACID MOLECULES,” filed Sep. 8, 2003, which claims thebenefit of priority under 35 U.S.C. 119(e) to U.S. provisionalapplication Ser. No. 60/457,135, entitled “RATIONAL EVOLUTION OFCYTOKINES FOR HIGHER STABILITY, ENCODING NUCLEIC ACID MOLECULES ANDRELATED APPLICATIONS,” filed Mar. 21, 2003, and U.S. provisionalapplication Ser. No. 60/409,898, entitled “RATIONAL EVOLUTION OFCYTOKINES FOR HIGHER STABILITY, ENCODING NUCLEIC ACID MOLECULES ANDRELATED APPLICATIONS,” filed Sep. 9, 2002; each to Rene Gantier, ThierryGuyon, Manuel Vega and Lila Drittanti.

This application is related to U.S. application Ser. No. 11/176,830,filed Jul. 6, 2005, which is also a continuation of U.S. applicationSer. No. 10/658,834, now U.S. Pat. No. 7,611,700, filed Sep. 8, 2003.This application is also related to PCT Application No. PCT/IB03/004347,entitled, “RATIONAL EVOLUTION OF CYTOKINES FOR HIGHER STABILITY, THECYTOKINES AND ENCODING NUCLEIC ACID MOLECULES,” to Rene Gantier, ThierryGuyon, Manuel Vega and Lila Drittanti. This application also is relatedto U.S. application Ser. No. 10/658,355, filed Sep. 8, 2003, entitled“RATIONAL DIRECTED PROTEIN EVOLUTION USING TWO-DIMENSIONAL RATIONALMUTAGENESIS SCANNING,” and to U.S. provisional application Ser. No.60/457,063, entitled “RATIONAL DIRECTED PROTEIN EVOLUTION USINGTWO-DIMENSIONAL RATIONAL MUTAGENESIS SCANNING,” filed Mar. 21, 2003, andto U.S. provisional application Ser. No. 60/410,258, entitled “RATIONALDIRECTED PROTEIN EVOLUTION USING TWO-DIMENSIONAL RATIONAL MUTAGENESISSCANNING,” filed Sep. 9, 2002, each to Rene Gantier, Thierry Guyon, HugoCruz Ramos, Manuel Vega and Lila Drittanti. This application also isrelated to U.S. application Ser. No. 10/022,249, now U.S. Pat. No.7,647,184, filed Dec. 17, 2001, entitled “HIGH THROUGHPUT DIRECTEDEVOLUTION BY RATIONAL MUTAGENESIS,” to Manuel Vega and Lila Drittanti.

The subject matter of each of the above-noted applications,international applications and provisional applications is incorporatedby reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ON COMPACT DISCS

An electronic version on compact disc (CD-R) of the Sequence Listing isfiled herewith in duplicate (labeled Copy #1 and Copy #2), the contentsof which are incorporated by reference in their entirety. Thecomputer-readable file on each of the aforementioned compact discs,created on Apr. 9, 2008 is identical, 1,843 kilobytes in size, andtitled 922JSEQ.001.txt.

FIELD OF INVENTION

Modified cytokine proteins having selected modified properties comparedto the unmodified or wild type proteins, and nucleic acid moleculesencoding these proteins are provided. The proteins can be used fortreatment and diagnosis.

BACKGROUND

The delivery of therapeutic proteins for clinical use is a majorchallenge to pharmaceutical science. Once in the blood stream, theseproteins are constantly eliminated from circulation within a short timeby different physiological processes, involving metabolism as well asclearance using normal pathways for protein elimination, such as(glomerular) filtration in the kidneys or proteolysis in blood. Thelatter is often the limiting process affecting the half-life of proteinsused as therapeutic agents in per-oral administration and eitherintravenous or intramuscular injection. The problems associated withthese routes of administration of proteins are well known and variousstrategies have been used in attempts to solve them.

A protein family, which has been the focus of much clinical work, andefforts to improve its administration and bio-assimilation, is thecytokine family, including the interferon family. Interferon moleculesare grouped in the heterogeneous family of cytokines, originallyidentified on the basis of their ability to induce cellular resistanceto viral infections (Diaz et al., J. Interferon Cytokine Res.,16:179-180, 1996). Type I interferons, referred to as interferons α/β,include many members of the interferon α family (interferon α1, α2, ωand τ) as well as interferon β. The type II interferon γ is differentfrom type I in its particular mechanisms that regulate its production.Whereas the production of interferons α/β is most efficiently induced inmany types of cells upon viral infection, interferon-γ is producedmainly in cells of hemopoietic system, such as T-cells or natural killercells, upon stimulation by antigens or cytokines, respectively. Thesetwo interferon systems are functionally non-redundant in the antiviraldefense host.

Interferon α, hereinafter “interferon alpha-2b,” or “interferon α-2b” or“IFNα-2b,” used interchangeably, has a broad spectrum of biologicaleffects, including antiviral effects. Antiviral effects includeantiproliferative and immuno-modulatory actions (Stark et al., Annu.Rev. Biochem., 67: 227-264, 1998). As well as eliciting strong antiviralactivities in target cells, interferons α/β also activate effector cellsof the innate immune system such as natural killer cells and macrophages(Pestka et al., Annu. Rev. Biochem., 56: 727-777, 1987; Biron et al.,Annu. Rev. Immunol., 17:189-220, 1999). As part of its immuno-modulatoryaction, interferon type I protects T-lymphocytes from apoptosis(Scheel-Toeller et al., Eur. J. Immunol., 29:2603-2612, 1999; Marrack etal., J. Exp. Med., 189:521-530, 1999) and growth enhancing factors(Robert et al., Hematol. Oncol., 4:113-120, 1986; Morikawa et al., J.Immunol., 139:761-766, 1987). The biological effects of interferons α/βare initiated upon binding to the IFN type I receptor, which results inactivation of several downstream effector molecules (Hibbert and Foster,J. Interferon Cytokine Res., 19:309-318, 1999).

Interferons as well as many cytokines are important therapeutics. Sincenaturally occurring variants have not evolved as therapeutics, theyoften have undesirable side-effects as well as the above-noted problemsof short-half life, administration and bioavailability. Hence, there isa need to improve properties of cytokines, including interferons, foruse as therapeutic agents. Therefore, among the objects herein, it is anobject to provide cytokines that have improved therapeutic properties.

SUMMARY

Provided herein are methods for directed evolution of families ofproteins and resulting families of modified proteins. A family, such asthe cytokine protein family, is initially identified. A property orphenotype for modification, such as resistance to proteolysis forincreased stability in blood, is selected for modification. Arepresentative member or members of the family, such as members of theinterferon α family, such as IFNα-2b or IFNα-2a, or interferon β family,is (are) selected. It is modified using any directed evolution methodand protein(s) with a desired phenotype are screened and identified. Inaddition, the 3-dimensional structure of the protein can be mapped totopologically and spatially identify the loci that are modified toachieve the phenotypic change. 3-dimensional structures of other membersof the family are generated or obtained and compared with the modifiedfamily member. Loci in the other family members that correspond on theprotein to those modified in the original protein are identified andmodified. The resulting proteins can be tested to confirm that theyexhibit the modified phenotype.

Provided herein are methods for generating modified cytokines based onstructural homology (3D scanning). These methods are based on thespatial and topological structure; they are not based on theirunderlying sequences of amino acid residues. The methods are used foridentification of target sites for mutagenesis, particularly in familiesof target proteins. The targets are identified through comparison ofpatterns of protein backbone folding between and among structurallyrelated proteins. The methods are exemplified herein for cytokines.Families of the modified cytokines also are provided herein.

Any protein known or otherwise available to those of skill in the art issuitable for modification, such as optimization or improvement of aselected property, using the directed evolution methods provided herein,including cytokines (e.g., IFNα, including IFNα-2b and IFNα-2a, andIFNβ) or any other proteins that have already been mutated or optimized.

Provided herein are modified cytokines that exhibit increased resistanceto proteolysis as assessed in vivo or in vitro. Typically the increasein resistance is a least 5%, generally 8%, 10% or more. The modifiedcytokines provided herein include those designed by 3D scanning usingthe interferon α's that were modified based upon 2D scanning methodsherein.

Also provided herein are modified (mutant) cytokine proteins, such asvariants of IFNβ and IFNα, including IFNα-2b and IFNα-2a proteins andIFNβ proteins, that have altered, particularly, improved therapeuticproperties, including higher stability compared to the unmodified forms.In particular, exemplary modified cytokines provided herein haveincreased stability, which, for example, improves their use astherapeutics. Among the modified cytokines provided herein are thosethat exhibit increased resistance to proteolysis compared to theunmodified cytokine. In particular, such resistance is at least 10%,20%, 30%, 40%, 50%, 70%, 100% or more resistant to proteolysis comparedto the unmodified cytokine. Also provided are cytokines that haveincreased anti-proliferative and/or antiviral activity and/or resistanceto proteolysis compared to an unmodified cytokine.

Exemplary of the modified cytokines provided herein are modifiedinterferons that exhibit higher stability compared to unmodified forms.Such modified interferons can be used for treating conditions in humansthat are responsive to treatment with interferons, such, but are notlimited to, as viral infections, cancer or tumors, undesired cellproliferation and for immuno-modulation.

Exemplary of proteins that can be modified by the 2D and 3D scanningmethods provided herein are cytokines from theinterferons/interleukin-10 family. This family includes, for example,interleukin-10 (IL-10; SEQ ID NO:200, interferon beta (IFNβ; SEQ ID NO:196), interferon alpha-2a (IFNα-2a; SEQ ID NO: 182), interferon alpha-2b(IFNα-2b; SEQ ID NO:1), and interferon gamma (IFN-γ, SEQ ID NO: 199).The long-chain cytokine protein family includes, among others,granulocyte colony stimulating factor (G-CSF; SEQ ID NO: 210), leukemiainhibitory factor (LIF; SEQ ID NO: 213), growth hormone (hGH; SEQ ID NO:216), ciliary neurotrophic factor (CNTF; SEQ ID NO: 212), leptin (SEQ IDNO: 211), oncostatin M (SEQ ID NO: 214), interleukin-6 (IL-6; SEQ ID NO:217) and interleukin-12 (IL-12; SEQ ID NO: 215). The short-chaincytokine protein family includes, among others, erythropoietin (EPO; SEQID NO: 201), granulocyte-macrophage colony stimulating factor (GM-CSF;SEQ ID NO: 202), interleukin-2 (IL-2; SEQ ID NO: 204), interleukin-3(IL-3; SEQ ID NO: 205), interleukin-4 (IL-4; SEQ ID NO: 207),interleukin-5 (IL-5; SEQ ID NO: 208), interleukin-13 (IL-13; SEQ ID NO:209), Flt3 ligand (SEQ ID NO: 203) and stem cell factor (SCF; SEQ ID NO:206). Modified forms of each that have increased resistance toproteolysis are provided. They were generated by comparison among the3D-structures to identify residues that improve resistance toproteolysis.

Pharmaceutical compositions containing each modified cytokine and usesand methods of treatment are provided.

The modified cytokines have use as therapeutics. Each cytokine hasimproved biological and or therapeutic activity, compared to the knowactivity of the unmodified cytokine. Accordingly, uses of the cytokinesfor treatment of cytokine-mediated diseases and diseases for whichimmunotherapy is employed are provided. Methods of treatment using themodified cytokines for diseases also are provided. Each cytokine has aknown therapeutic use, and such use is contemplated herein. Cytokinesprovided herein have improved properties, such as increasedbioavailability, improved stability, particularly in vivo, and/orgreater efficacy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(A) displays the sequence of the mature IFNα-2b (SEQ ID NO:1).Residues targeted by a mixture of proteases, including α-chymotrypsin(F, L, M, W, and Y), endoproteinase Arg-C(R), endoproteinase Asp-N (D),endoproteinase Glu-C (E), endoproteinase Lys-C (K), and trypsin (K, andR), are underlined and in bold lettering.

FIG. 1(B) displays the structure of IFNα-2b obtained from the NMRstructure of IFNα-2a (PDB code 1ITF) in ribbon representation. Surfaceresidues exposed to the action of the proteases considered in FIG. 1Aare in space filling representation.

FIG. 2 depicts the “Percent Accepted Mutation” (PAM250) matrix Valuesgiven to identical residues are shown in gray squares. Highest values inthe matrix are shown in black squares and correspond to the highestoccurrence of substitution between two residues.

FIG. 3 presents the scores obtained from PAM250 analysis for the aminoacid substitutions (replacing amino acids on the vertical axis; aminoacid position on the horizontal axis) aimed at introducing resistance toproteolysis into the IFNα-2b at the protease target sequences. The twobest replacing residues for each target amino acid according to thehighest substitution scores are shown in black rectangles.

FIGS. 4(A)-4(C) provide graphs of experiments indicating the levels ofprotection against in vitro proteolysis for IFNα-2b variants produced inmammalian cells. In FIGS. 4(B) and 4(C), the vertical axis indicates therelative level of non-proteolyzed protein and the horizontal axisindicates time in hours.

FIG. 5 displays the characterization of several IFNα-2b variants,produced in mammalian cells, treated with α-chymotrypsin.

FIG. 6(A) shows the characterization of the E113H IFNα-2b variant whentreated with α-chymotrypsin. The percent of residual (anti-viral)activity for the variant (black line and white circles) after treatmentwith α-chymotrypsin was compared to the treated wild-type IFNα-2b(dashed line and black squares). For this experiment, the E113H IFNα-2bvariant was produced in mammalian cells.

FIG. 6(B) shows the characterization of the E113H IFNα-2b varianttreated with a mixture of proteases. The percent of residual(anti-viral) activity for the variant (black line and white circles)after treatment with protease mixture was compared to the treatedwild-type IFNα-2b (dashed line and black squares). For this experiment,the E113H IFNα-2b variant was produced in mammalian cells.

FIG. 6(C) presents the characterization of the E113H IFNα-2b varianttreated with blood lysate. The percent of residual (anti-viral) activityfor the variant (black line and white circles) after treatment withblood lysate was compared to the treated wild-type IFNα-2b (dashed lineand black squares). For this experiment, the E113H IFNα-2b variant wasproduced in mammalian cells.

FIG. 6(D) presents the characterization of the E113H IFNα-2b varianttreated with serum. The percent of residual (anti-viral) activity forthe variant (black line and white circles) after treatment with serumwas compared to the treated wild-type IFNα-2b (dashed line and blacksquares). For this experiment, the E113H IFNα-2b variant was produced inmammalian cells.

FIGS. 6(E) and 6(F) provide graphs indicating the levels of protectionagainst in vitro proteolysis for IFNα-2b variants produced in bacteria.In FIGS. 6(E) and 6(F), the vertical axis indicates the relative levelof non-proteolyzed protein and the horizontal axis indicates time inhours. The percent of residual (anti-viral) activity for the variants(gray circles with continuous lines) after treatment were compared tothe treated wild-type IFNα-2b (solid circles with dashed lines).

FIG. 6(G) provides graphs indicating the in vitro potency for antiviralactivity, for IFNα-2b variants produced in bacteria. The vertical axisindicates the level of antiviral activity and the horizontal axisindicates concentration of the variants at which each level of activityis achieved. The activity for the variants (continuous line with graycircles) was compared to that of the wild-type IFNα-2b (black triangleswith dashed lines). The potency for each variant was calculated from thegraphs as the concentration at the inflection point of the respectivecurves. FIG. 6(T) shows the value of potency obtained for each varianttested compared to the wild type IFNα.

FIG. 6(H) provides the in vitro potency for anti-proliferation activity,for IFNα-2b variants produced in bacteria. The activity for the variantswas compared to that of the wild-type IFNα-2b in serial dilutionexperiments where the anti-proliferation activity was measured for anumber of dilutions for each variant. Potency was calculated from thegraphs as the concentration at the inflection point of the respectivecurves. The figure shows the value of potency obtained for each varianttested and in comparison to the wild type IFNα.

FIGS. 6(I) to 6(N) provide graphs indicating the pharmacokinetics inmice following subcutaneous injection of IFNα-2b variants produced inbacteria. The vertical axis indicates the level of antiviral activity inblood and the horizontal axis indicates the time after injection atwhich the level of antiviral activity is determined. Thepharmacokinetics of the variants (in gray solid circles with graycontinuous lines) was compared to that of the wild-type IFNα-2b (inblack with dashed lines) and of a pegylated derivative (Pegasys, Roche)(36 μg/ml open triangles with continuous black lines; and 18 μg/ml opencircles with continuous black lines); and vehicle (gray solid triangleswith continuous gray lines. The Area Under the Curve (AUC) for eachvariant was calculated from the graphs and is shown in 6(U).

FIG. 6(O) provides graphs indicating the levels of protection against invitro proteolysis for IFNβ variants produced in mammalian cells. FIG.6(N), the vertical axis indicates the relative level of non-proteolyzedprotein and the horizontal axis indicates time in hours. The percent ofresidual (anti-viral) activity for the variants after treatment werecompared to the treated wild-type IFNβ.

FIGS. 6(P) to 6(S) provide graphs indicating the in vitro potency foreither antiviral activity (6(P) and 6(Q)) or anti-proliferative activity(6(R) and 6(S), for a number of IFNβ variants produced in mammaliancells. The vertical axis indicates the level of (antiviral oranti-proliferation) activity and the horizontal axis indicates theconcentration of the variants at which each level of activity isachieved. The activity for the variants (6(Q) and (6(S)) was compared tothat of the wild-type IFNβ (6(P) and (6(R)). The activity obtained witheither no previous treatment or by treating the variants with proteasesprior to the activity test is shown.

FIG. 6(T) provides a comparison of antiviral activity (potency),anti-proliferation activity (potency), number of mutations present andAUC (from PK) for a number of IFNα-2b and in comparison with thewild-type IFNα-2b.

FIG. 6(U) provides IFN units injected and protein injected (μg/ml) forthe data in FIG. 6(T).

FIG. 7(A) depicts a top view ribbon representation of IFNα-2b structureobtained from the NMR structure of IFNα-2a (PDB code 1ITF). Residuesrepresented in “space filling” define (1) the “receptor binding region”based on either our “alanine scanning” analysis or on studies by Piehleret al., J. Biol. Chem., 275:40425-40433, 2000, and Roisman et al., Proc.Natl. Acad. Sci. USA, 98:13231-13236, 2001 (in light-gray and dark-gray,respectively), and (2) replacing residues (LEADs) for resistance toproteolysis (in black).

FIG. 7(B) depicts a side view ribbon representation of IFNα-2bstructure. Residue representation is as in FIG. 7A.

FIG. 8(A) schematizes the identification of homologous amino acidpositions between a number of cytokines and the LEAD mutants of IFNα-2busing 3-dimensional scanning (also referred to herein as based on“structure-based homology” methods or “structural homology” methods).

FIG. 8(B) illustrates a structural overlapping between human interferonα-2b obtained from the NMR structure of IFNα-2a (PDB code 1ITF) andhuman interferon β (PDB code 1AU1) using Swiss PDB Viewer.

FIG. 8(C) illustrates a structural overlapping between human interferonα-2b obtained from the NMR structure of IFNα-2a (PDB code 1ITF) anderythropoietin (PDB code 1BUY) using Swiss PDB Viewer.

FIG. 8(D) illustrates a structural overlapping between human interferonα-2b obtained from the NMR structure of IFNα-2a (PDB code 1ITF) andgranulocyte-colony stimulating factor (PDB code 1CD9) using Swiss PDBViewer.

FIG. 9 illustrates a structural alignment of a number of cytokines andinterferon α-2b sequences (SEQ ID NO: 1 (IFN-α-2b); SEQ ID NO: 196(IFN-β); SEQ ID NO: 201 (EPO); and SEQ ID NO: 210 (G-CSF)). Boldunderlined residues define the region on each cytokine sequence thatbased on structural homology comparison corresponds to thestructurally-related mutations found on the LEADs for proteaseresistance of IFNα-2b.

FIG. 10(A) shows the antiviral activity of interferon α-2b mutantsgenerated by alanine-scanning analysis used for protein redesign.Plotted symbols for wild type and variants of interferon α-2b areindicated in the inset.

FIG. 10(B) displays cell proliferation after treatment with interferonα-2b mutants obtained by alanine-scanning analysis. Plotted symbols forwild type and variants of interferon α-2b are indicated in the inset.

FIG. 10(C) displays the correlation between the antiviral activity andcell proliferation activity of interferon α-2b mutants obtained byalanine-scanning analysis.

FIG. 11 Candidate glycosylation sites for interferon α-2b stabilizationand redesign thereof.

FIG. 12 (A) shows a representative number of the is-HIT residuepositions and type of replacing amino acids selected to generatemodified protein sequences of interferon β (corresponding to SEQ ID Nos:233-289, 989-1015, and 1016-1302) compared to the wild-type sequence(SEQ ID NO: 196), based on 3D-scanning (structural homology method),including PAM250 analysis.

FIG. 12 (B) displays the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences ofinterferon gamma (corresponding to SEQ ID Nos: 290-311) compared toresidues 1-100 of the wild-type sequence (SEQ ID NO: 199), based onstructural homology and PAM250 analysis.

FIG. 12 (C) shows the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences ofinterleukin-10 (corresponding to SEQ ID Nos: 312-361) compared toresidues 1-100 of the wild-type sequence (SEQ ID NO: 200), based onstructural homology and PAM250 analysis.

FIG. 12 (D) displays the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences of ciliaryneurotrophic factor (corresponding to SEQ ID Nos: 684-728) compared toresidues 51-188 of the wild-type sequence (SEQ ID NO: 212), based onstructural homology and PAM250 analysis.

FIG. 12 (E) shows the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences ofgranulocyte-colony stimulating factor (corresponding to SEQ ID Nos:631-662) compared to residues 51-177 of the wild-type sequence (SEQ IDNO: 210), based on structural homology and PAM250 analysis.

FIG. 12 (F) displays the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences of humangrowth hormone (corresponding to SEQ ID Nos: 850-895) compared toresidues 51-191 of the wild-type sequence (SEQ ID NO: 216), based onstructural homology and PAM250 analysis.

FIG. 12 (G) shows the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences ofinterleukin-12 (corresponding to SEQ ID Nos: 794-849) compared toresidues 51-197 of the wild-type sequence (SEQ ID NO: 215), based onstructural homology and PAM250 analysis.

FIG. 12 (H) displays the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences ofinterleukin-6 (corresponding to SEQ ID Nos: 896-939) compared toresidues 51-183 of the wild-type sequence (SEQ ID NO: 217), based onstructural homology and PAM250 analysis.

FIG. 12 (I) shows the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences of leptin(corresponding to SEQ ID Nos: 663-683) compared to the wild-typesequence (SEQ ID NO: 211), based on structural homology and PAM250analysis.

FIG. 12 (J) displays the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences of leukemiainhibitory factor (corresponding to SEQ ID Nos: 729-760) compared toresidues 51-180 of the wild-type sequence (SEQ ID NO: 213), based onstructural homology and PAM250 analysis.

FIG. 12 (K) shows the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences ofoncostatin M (corresponding to SEQ ID Nos: 761-793) compared to residues51-150 of the wild-type sequence (SEQ ID NO: 214), based on structuralhomology and PAM250 analysis.

FIG. 12 (L) displays the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences oferythropoietin (corresponding to SEQ ID Nos: 940-977) compared to thewild-type sequence (SEQ ID NO: 201), based on structural homology andPAM250 analysis.

FIG. 12 (M) shows the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences of Flt3ligand (corresponding to SEQ ID Nos: 401-428) compared to residues 1-100of the wild-type sequence (SEQ ID NO: 203), based on structural homologyand PAM250 analysis.

FIG. 12 (N) displays the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences ofgranulocyte-macrophage colony-stimulating factor (corresponding to SEQID Nos: 362-400) compared to the wild-type sequence (SEQ ID NO: 202),based on structural homology and PAM250 analysis.

FIG. 12 (O) shows the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences ofinterleukin-13 (corresponding to SEQ ID Nos: 603-630) compared to thewild-type sequence (SEQ ID NO: 209), based on structural homology andPAM250 analysis.

FIG. 12 (P) displays the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences ofinterleukin-2 (corresponding to SEQ ID Nos: 429-476) compared to thewild-type sequence (SEQ ID NO: 204), based on structural homology andPAM250 analysis.

FIG. 12 (Q) shows the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences ofinterleukin-3 (corresponding to SEQ ID Nos: 477-498) compared to thewild-type sequence (SEQ ID NO: 205), based on structural homology andPAM250 analysis.

FIG. 12 (R) displays the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences ofinterleukin-4 (corresponding to SEQ ID Nos: 543-567) compared to thewild-type sequence (SEQ ID NO: 207), based on structural homology andPAM250 analysis.

FIG. 12 (S) shows the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences ofinterleukin-5 (corresponding to SEQ ID Nos: 568-602) compared to thewild-type sequence (SEQ ID NO: 208), based on structural homology andPAM250 analysis.

FIG. 12 (T) displays the is-HIT residue positions and type of replacingamino acids selected to generate modified protein sequences of stem cellfactor (corresponding to SEQ ID Nos: 499-542) compared to residues 1-141of the wild-type sequence (SEQ ID NO: 206), based on structural homologyand PAM250 analysis.

DETAILED DESCRIPTION

A. Definitions

B. Directed Evolution

-   -   1. Pure Random Mutagenesis    -   2. Restricted Random Mutagenesis    -   3. Non-Restricted Rational Mutagenesis

C. 2-Dimensional Rational Scanning (2D Scanning)

-   -   1. Identifying In-silico HITS    -   2. Identifying Replacing Amino Acids        -   a. Percent Accepted Mutation (PAM)            -   i. PAM Analysis            -   ii. PAM250        -   b. Jones et al. and Gonnet et al.        -   c. Fitch and Feng et al.        -   d. McLachlan, Grantham and Miyata        -   e. Rao        -   f. Risler et al.        -   g. Johnson et al.        -   h. Block Substitution Matrix (BLOSUM)    -   3. Physical Construction of Mutant Proteins and Biological        Assays

D. 2-Dimensional Scanning of Proteins for Increased Resistance toProteolysis

E. Rational Evolution of IFNα-2b For Increased Resistance to Proteolysis

-   -   1. Modified IFNα-2b and IFNα-2a Proteins with Single Amino Acid        Substitutions        -   (is-HITs)    -   2. LEAD identification    -   3. N-glycosylation Site Addition

F. Protein Redesign

G. 3D-scanning and Its Use for Modifying Cytokines

-   -   1. Homology    -   2. 3D-Scanning (Structural Homology) Methods    -   3. Application of the 3D-Scanning Method to Cytokines        -   a. Structurally Homologous Interferon Mutants        -   b. Structurally Homologous Cytokine Mutants

H. Rational Evolution of IFNβ For Increased Resistance to Proteolysisand/or Higher Conformational Stability

I. Super-LEADs and Additive Directional Mutagenesis (ADM).

-   -   1. Additive Directional Mutagenesis    -   2. Multi-Overlapped Primer Extensions

J. Uses of the Mutant IFNα and IFNβ Genes and Cytokines in TherapeuticMethods

-   -   1. Fusion Proteins    -   2. Nucleic Acid Molecules for Expression    -   3. Formulation of Optimized Cytokines and Methods of Treatment

K. Examples

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, Genbank sequences, websites andother published materials referred to throughout the entire disclosureherein, unless noted otherwise, are incorporated by reference in theirentirety. In the event that there is a plurality of definitions forterms herein, those in this section prevail. Where reference is made toa URL or other such identifier or address, it understood that suchidentifiers can change and particular information on the internet cancome and go, but equivalent information can be found by searching theinternet. Reference thereto evidences the availability and publicdissemination of such information.

As used herein, biological activity of a protein refers to any activitymanifested by the protein in vivo.

As used herein, “a directed evolution method” refers to methods that“adapt” either natural proteins, synthetic proteins or protein domainsto work in new or existing natural or artificial chemical or biologicalenvironments and/or to elicit new functions and/or to increase ordecrease a given activity, and/or to modulate a given feature. Exemplarydirected evolution methods include pure random mutagenesis methods;restricted random mutagenesis methods; and non-restricted rationalmutagenesis methods, such as the rational directed evolution methoddescribed in co-pending U.S. application Ser. No. 10/022,249; and the2-dimensional rational scanning method provided herein.

As used herein, two dimensional rational mutagenesis scanning (2Dscanning) refers to the processes provided herein in which twodimensions of a particular protein sequence are scanned: (1) onedimension is to identify specific amino acid residues along the proteinsequence to replace with different amino acids, referred to as is-HITtarget positions, and (2) the second dimension is the amino acid typeselected for replacing the particular is-HIT target, referred to as thereplacing amino acid.

As used herein, in silico refers to research and experiments performedusing a computer. In silico methods include, but are not limited to,molecular modeling studies, and biomolecular docking experiments.

As used herein, “is-HIT” refers to an in silico identified amino acidposition along a target protein sequence that has been identified basedon i) the particular protein properties to be evolved, ii) the protein'samino acid sequence, and/or iii) the known properties of the individualamino acids. These is-HIT loci on the protein sequence are identifiedwithout use of experimental biological methods. For example, once theprotein feature(s) to be optimized is (are) selected, diverse sources ofinformation or previous knowledge (i.e., protein primary, secondary ortertiary structures, literature, patents) are exploited to determinethose amino acid positions that may be amenable to improved proteinfitness by replacement with a different amino acid. This step utilizesprotein analysis “in silico.” All possible candidate amino acidpositions along a target protein's primary sequence that might beinvolved in the feature being evolved are referred to herein as “insilico HITs” (“is-HITs”). The collection (library), of all is-HITsidentified during this step represents the first dimension (targetresidue position) of the two-dimensional scanning methods providedherein.

As used herein, “amenable to providing the evolved predeterminedproperty or activity,” in the context of identifying is-HITs, refers toan amino acid position on a protein that is contemplated, based on insilico analysis, to possess properties or features that when replacedwould result in the desired activity being evolved. The phrase “amenableto providing the evolved predetermined property or activity,” in thecontext of identifying replacement amino acids, refers to a particularamino acid type that is contemplated, based on in silico analysis, topossess properties or features that when used to replace the originalamino acid in the unmodified starting protein would result in thedesired activity being evolved.

As used herein, high-throughput screening (HTS) refers to processes thattest a large number of samples, such as samples of test proteins orcells containing nucleic acids encoding the proteins of interest toidentify structures of interest or the identify test compounds thatinteract with the variant proteins or cells containing them. HTSoperations are amenable to automation and are typically computerized tohandle sample preparation, assay procedures and the subsequentprocessing of large volumes of data.

As used herein, the term “restricted,” when used in the context of theidentification of is-HIT amino acid positions along the protein sequenceselected for amino acid replacement and/or the identification ofreplacing amino acids, means that fewer than all amino acids on theprotein-backbone are selected for amino acid replacement; and/or fewerthan all of the remaining 19 amino acids available to replace theoriginal amino acid present in the unmodified starting protein areselected for replacement. In particular embodiments of the methodsprovided herein, the is-HIT amino acid positions are restricted, suchthat fewer than all amino acids on the protein-backbone are selected foramino acid replacement. In other embodiments, the replacing amino acidsare restricted, such that fewer than all of the remaining 19 amino acidsavailable to replace the native amino acid present in the unmodifiedstarting protein are selected as replacing amino acids. In a particularembodiment, both of the scans to identify is-HIT amino acid positionsand the replacing amino acids are restricted, such that fewer than allamino acids on the protein-backbone are selected for amino acidreplacement and fewer than all of the remaining 19 amino acids availableto replace the native amino acid are selected for replacement.

As used herein, “candidate LEADs,” are mutant proteins that arecontemplated as potentially having an alteration in any attribute,chemical, physical or biological property in which such alteration issought. In the methods herein, candidate LEADs are generally generatedby systematically replacing is-HITS loci in a protein or a domainthereof with typically a restricted subset, or all, of the remaining 19amino acids, such as obtained using PAM analysis. Candidate LEADs can begenerated by other methods known to those of skill in the art tested bythe high throughput methods herein.

As used herein, “LEADs” are “candidate LEADs” whose activity has beendemonstrated to be optimized or improved for the particular attribute,chemical, physical or biological property. For purposes herein a “LEAD”typically has activity with respect to the function of interest thatdiffers by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,150%, 200% or more from the unmodified and/or wild type (native)protein. In certain embodiments, the change in activity is at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, of theactivity of the unmodified target protein. In other embodiments, thechange in activity is not more than about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100%, of the activity of the unmodified target protein.In yet other embodiments, the change in activity is at least about 2times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times,600 times, 700 times, 800 times, 900 times, 1000 times, or more greaterthan the activity of the unmodified target protein. The desiredalteration, which can be either an increase or a reduction in activity,will depend upon the function or property of interest (e.g., ±10%, ±20%,etc.). The LEADs may be further optimized by replacement of a plurality(2 or more) of “is-HIT” target positions on the same protein molecule togenerate “super-LEADs.”

As used herein, the term “super-LEAD” refers to protein mutants(variants) obtained by combining t he single mutations present in two ormore of the LEAD molecules into a single protein molecule. Accordingly,in the context of the modified proteins provided herein, the phrase“proteins comprising one or more single amino acid replacements”encompasses any combination of two or more of the mutations describedherein for a respective protein. For example, the modified proteinsprovided herein having one or more single amino acid replacements canhave can have any combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or more of the amino acid replacements at thedisclosed replacement positions. The collection of super-LEAD mutantmolecules is generated, tested and phenotypically characterizedone-by-one in addressable arrays. Super-LEAD mutant molecules are suchthat each molecule contains a variable number and type of LEADmutations. Those molecules displaying further improved fitness for theparticular feature being evolved, are referred to as super-LEADs.Super-LEADs can be generated by other methods known to those of skill inthe art and tested by the high throughput methods herein. For purposesherein a super-LEAD typically has activity with respect to the functionof interest that differs from the improved activity of a LEAD by adesired amount, such as at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 150%, 200% or more from at least one of the LEAD mutants fromwhich it is derived. As with LEADs, the change in the activity forsuper-LEADs is dependent upon the activity that is being “evolved.” Thedesired alteration, which can be either an increase or a reduction inactivity, will depend upon the function or property of interest.

As used herein, a recitation that modified protein has more antiviralactivity (or other activity) than antiproliferative activity (or anotheractivity) compared to the unmodified cytokine, is comparing the absolutevalue of the change in each activity compared to wild type.

As used herein, the phrase “altered loci” refers to the is-HIT aminoacid positions in the LEADs or super-LEADs that are replaced withdifferent replacing amino acids, resulting in the desired alteredphenotype or activity.

As used herein, an exposed residue presents more than 15% of its surfaceexposed to the solvent.

As used herein, the phrase “structural homology” refers to the degree ofcoincidence in space between two or more protein backbones. Proteinbackbones that adopt the same protein structure, fold and showsimilarity upon three-dimensional structural superposition in space canbe considered structurally homologous. Structural homology is not basedon sequence homology, but rather on three-dimension homology. Two aminoacids in two different proteins said to be homologous based onstructural homology between those proteins, do not necessarily need tobe in sequence-based homologous regions. For example, protein backbonesthat have a root mean squared (RMS) deviation of less than 3.5, 3.0,2.5, 2.0, 1.7 or 1.5 angstroms (Å) at a given space position or definedregion between each other can be considered to be structurallyhomologous in that region, and are referred to herein as having a “highcoincidence” between their backbones. It is contemplated herein thatsubstantially equivalent (e.g., “structurally related”) amino acidpositions that are located on two or more different protein sequencesthat share a certain degree of structural homology will have comparablefunctional tasks; also referred to herein as “structurally homologousloci.” These two amino acids than can be said to be “structurallysimilar” or “structurally related” with each other, even if theirprecise primary linear positions on the amino acid sequences, when thesesequences are aligned, do not match with each other. Amino acids thatare “structurally related” can be far away from each other in theprimary protein sequences, when these sequences are aligned followingthe rules of classical sequence homology.

As used herein, a structural homolog is a protein that is generated bystructural homology.

As used herein, the phrase “unmodified target protein,” “unmodifiedprotein” or “unmodified cytokine,” or grammatical variations thereof,refers to a starting protein that is selected for modification using themethods provided herein. The starting unmodified target protein can bethe naturally occurring, wild type form of a protein. In addition, thestarting unmodified target protein may have previously been altered ormutated, such that it differs from the native wild type isoform, but isnonetheless referred to herein as a starting unmodified target proteinrelative to the subsequently modified proteins produced herein. Thus,existing proteins known in the art that have previously been modified tohave a desired increase or decrease in a particular biological activitycompared to an unmodified reference protein can be selected and usedherein as the starting “unmodified target protein.” For example, aprotein that has been modified from its native form by one or moresingle amino acid changes and possesses either an increase or decreasein a desired activity, such as resistance to proteolysis, can beutilized with the methods provided herein as the starting unmodifiedtarget protein for further modification of either the same or adifferent biological activity.

Likewise, existing proteins known in the art that have previously beenmodified to have a desired increase or decrease in a particularbiological activity compared to an unmodified reference protein can beselected and used herein for identification of structurally homologousloci on other structurally homologous target proteins. For example, aprotein that has been modified by one or more single amino acid changesand possesses either an increase or decrease in a desired activity, suchas resistance to proteolysis, can be utilized with the methods providedherein to identify on structurally homologous target proteins,corresponding structurally homologous loci that can be replaced withsuitable replacing amino acids and tested for either an increase ordecrease in the desired biological activity.

As used herein, the phrase “only one amino acid replacement occurs oneach target protein” refers to the modification of a target protein,such that it differs from the unmodified form of the target protein by asingle amino acid change. For example, in one embodiment, mutagenesis isperformed by the replacement of a single amino acid residue at only oneis-HIT target position on the protein backbone (e.g., “one-by-one” inaddressable arrays), such that each individual mutant generated is thesingle product of each single mutagenesis reaction. The single aminoacid replacement mutagenesis reactions are repeated for each of thereplacing amino acids selected at each of the is-HIT target positions.Thus, a plurality of mutant protein molecules are produced, whereby eachmutant protein contains a single amino acid replacement at only one ofthe is-HIT target positions.

As used herein, the phrase “pseudo-wild type,” in the context of singleor multiple amino acid replacements, are those amino acids that, whiledifferent from the original, such as native, amino acid at a given aminoacid position, can replace the native one at that position withoutintroducing any measurable change in a particular protein activity. Apopulation of sets of nucleic acid molecules encoding a collection ofmutant molecules is generated and phenotypically characterized such thatproteins with amino acid sequences different from the original aminoacid, but that still elicit substantially the same level (i.e., at least10%, 50%, 70%, 90%, 95%, 100%, depending upon the protein) and type ofdesired activity as the original protein are selected.

As used herein, biological and pharmacological activity includes anyactivity of a biological pharmaceutical agent and includes, but is notlimited to, resistance to proteolysis, biological efficiency,transduction efficiency, gene/transgene expression, differential geneexpression and induction activity, titer, progeny productivity,toxicity, cytotoxicity, immunogenicity, cell proliferation and/ordifferentiation activity, anti-viral activity, morphogenetic activity,teratogenetic activity, pathogenetic activity, therapeutic activity,tumor suppressor activity, ontogenetic activity, oncogenetic activity,enzymatic activity, pharmacological activity, cell/tissue tropism anddelivery.

As used herein, a “small region” on a polypeptide is relative termdepending upon the size of the polypeptide, but typically refers to aregion that is less than about 10%, 15%, 25% of the protein. A largeregion is greater than about 10%, 15% or 25% of the protein.

As used herein, “output signal” refers to parameters that can befollowed over time and, if desired, quantified. For example, when arecombinant protein is introduced into a cell, the cell containing therecombinant protein undergoes a number of changes. Any such change thatcan be monitored and used to assess the transformation or transfection,is an output signal, and the cell is referred to as a reporter cell; theencoding nucleic acid is referred to as a reporter gene, and theconstruct that includes the encoding nucleic acid is a reporterconstruct. Output signals include, but are not limited to, enzymeactivity, fluorescence, luminescence, amount of product produced andother such signals. Output signals include expression of a gene or geneproduct, including heterologous genes (transgenes) inserted into theplasmid virus. Output signals are a function of time (“t”) and arerelated to the amount of protein used in the composition. For higherconcentrations of protein, the output signal can be higher or lower. Forany particular concentration, the output signal increases as a functionof time until a plateau is reached. Output signals can also measure theinteraction between cells, expressing heterologous genes, and biologicalagents

As used herein, the activity of an IFNα-2b or IFNα-2a protein refers toany biological activity that can be assessed. In particular, herein, theactivity assessed for the IFNα-2b or IFNα-2a proteins is resistance toproteolysis, antiviral activity and cell proliferation activity.

As used herein, the Hill equation is a mathematical model that relatesthe concentration of a drug (i.e., test compound or substance) to theresponse measured

$y = \frac{{y_{\max}\lbrack D\rbrack}^{x}}{\lbrack D\rbrack^{n} + \left\lbrack D_{50} \right\rbrack^{n}}$where y is the variable measured, such as a response, signal, y_(max) isthe maximal response achievable, [D] is the molar concentration of adrug, [D50] is the concentration that produces a 50% maximal response tothe drug, n is the slope parameter, which is 1 if the drug binds to asingle site and with no cooperativity between or among sites. A Hillplot is log₁₀ of the ratio of ligand-occupied receptor to free receptorvs. log [D] (M). The slope is n, where a slope of greater than 1indicates cooperativity among binding sites, and a slope of less than 1can indicate heterogeneity of binding. This general equation has beenemployed for assessing interactions in complex biological systems (see,published International PCT application No. WO 01/44809 based on PCT No.PCT/FR00/03503, see also, the EXAMPLES).

As used herein, in the Hill-based analysis (published International PCTapplication No. WO 01/44809 based on PCT No. PCT/FR00/03503), theparameters, π, κ, τ, ε, η, θ, are as follows:

π is the potency of the biological agent acting on the assay(cell-based) system;

κ is the constant of resistance of the assay system to elicit a responseto a biological agent;

ε is the global efficiency of the process or reaction triggered by thebiological agent on the assay system;

τ is the apparent titer of the biological agent;

θ is the absolute titer of the biological agent; and

η is the heterogeneity of the biological process or reaction.

In particular, as used herein, the parameters π (potency) or κ (constantof resistance) are used to respectively assess the potency of a testagent to produce a response in an assay system and the resistance of theassay system to respond to the agent.

As used herein, ε (efficiency), is the slope at the inflexion point ofthe Hill curve (or, in general, of any other sigmoidal or linearapproximation), to assess the efficiency of the global reaction (thebiological agent and the assay system taken together) to elicit thebiological or pharmacological response.

As used herein, τ (apparent titer) is used to measure the limitingdilution or the apparent titer of the biological agent.

As used herein, θ (absolute titer), is used to measure the absolutelimiting dilution or titer of the biological agent.

As used herein, η (heterogeneity) measures the existence ofdiscontinuous phases along the global reaction, which is reflected by anabrupt change in the value of the Hill coefficient or in the constant ofresistance.

As used herein, a population of sets of nucleic acid molecules encodinga collection (library) of mutants refers to a collection of plasmids orother vehicles that carry (encode) the gene variants, such thatindividual plasmids or other individual vehicles carry individual genevariants. Each element (member) of the collection is physicallyseparated from the others, such as individually in an appropriateaddressable array, and has been generated as the single product of anindependent mutagenesis reaction. When a collection (library) of suchproteins is contemplated, it will be so-stated.

As used herein, a “reporter cell” is the cell that “reports,” i.e.,undergoes the change, in response to a condition, such as, for example,exposure to a protein or a virus or to a change it its external orinternal environment.

As used herein, “reporter” or “reporter moiety” refers to any moietythat allows for the detection of a molecule of interest, such as aprotein expressed by a cell. Reporter moieties include, but are notlimited to, for example, fluorescent proteins, such as red, blue andgreen fluorescent proteins; LacZ and other detectable proteins and geneproducts. For expression in cells, nucleic acid encoding the reportermoiety can be expressed as a fusion protein with a protein of interestor under to the control of a promoter of interest.

As used herein, phenotype refers to the physical, physiological or othermanifestation of a genotype (a sequence of a gene). In methods herein,phenotypes that result from alteration of a genotype are assessed.

As used herein, “activity” means in the largest sense of the term anychange in a system (either biological, chemical or physical system) ofany nature (changes in the amount of product in an enzymatic reaction,changes in cell proliferation, in immunogenicity, in toxicity) caused bya protein or protein mutant when they interact with that system. Inaddition, the term “activity,” “higher activity” or “lower activity” asused herein in reference to resistance to proteases, proteolysis,incubation with serum or with blood, means the ratio or residualbiological (antiviral) activity between “after” protease/blood or serumtreatment and “before” protease/blood or serum treatment.

As used herein, activity refers to the function or property to beevolved. An active site refers to a site(s) responsible or thatparticipates in conferring the activity or function. The activity oractive site evolved (the function or property and the site conferring orparticipating in conferring the activity) can have nothing to do withnatural activities of a protein. For example, it could be an “activesite” for conferring immunogenicity (immunogenic sites or epitopes) on aprotein.

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease are ameliorated or otherwise beneficiallyaltered. Treatment also encompasses any pharmaceutical use of themodified cytokines and compositions provided herein.

As used herein, cytokine-mediated or cytokine-involved diseases refer todiseases in which cytokines potentiate, cause or are involved in thedisease process or to diseases in which administration of a cytokine isameliorative of a disease or symptoms thereof. Cytokines can be used inimmunotherapeutic therapies or protocols.

As used herein, the amino acids, which occur in the various amino acidsequences appearing herein, are identified according to their known,three-letter or one-letter abbreviations (see, Table 1). Thenucleotides, which occur in the various nucleic acid fragments aredesignated with the standard single-letter designations used routinelyin the art.

As used herein, amino acid residue refers to an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are presumed to be inthe “L” isomeric form. Residues in the “D” isomeric form, which areso-designated, can be substituted for any L-amino acid residue, as longas the desired functional property is retained by the polypeptide. NH₂refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxy group present at thecarboxyl terminus of a polypeptide. In keeping with standard polypeptidenomenclature described in J. Biol. Chem., 243:3552-3559, 1969, andadopted at 37 C.F.R. §§1.821-1.822, abbreviations for amino acidresidues are shown in Table 1:

TABLE 1 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr tyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp tryptophan R Arg arginine DAsp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine XXaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by formulae have a left to right orientation in the conventionaldirection of amino-terminus to carboxyl-terminus. In addition, thephrase “amino acid residue” is broadly defined to include the aminoacids listed in the Table of Correspondence (Table 1) and modified andunusual amino acids, such as those referred to in 37 C.F.R.§§1.821-1.822, and incorporated herein by reference. Furthermore, itshould be noted that a dash at the beginning or end of an amino acidresidue sequence indicates a peptide bond to a further sequence of oneor more amino acid residues or to an amino-terminal group such as NH2 orto a carboxyl-terminal group such as COOH.

As used herein, nucleic acids include DNA, RNA and analogs thereof,including protein nucleic acids (PNA) and mixtures thereof. Nucleicacids can be single or double stranded. When referring to probes orprimers, optionally labeled, with a detectable label, such as afluorescent or radiolabel, single-stranded molecules are contemplated.Such molecules are typically of a length such that they arestatistically unique of low copy number (typically less than 5,generally less than 3) for probing or priming a library. Generally aprobe or primer contains at least 14, 16 or 30 contiguous of sequencecomplementary to or identical a gene of interest. Probes and primers canbe 10, 14, 16, 20, 30, 50, 100 or more nucleic acid bases long.

Therefore, as used herein, the term “identity” represents a comparisonbetween a test and a reference polypeptide or polynucleotide. Forexample, a test polypeptide can be defined as any polypeptide that is90% or more identical to a reference polypeptide.

As used herein, “corresponding structurally-related” positions on two ormore proteins, such as the IFNα-2b protein and other cytokines, refersthose amino acid positions determined based upon structural homology tomaximize tri-dimensional overlapping between proteins.

As used herein, the term at least “90% identical to” refers to percentidentities from 90 to 100% relative to the reference polypeptides.Identity at a level of 90% or more is indicative of the fact that,assuming for exemplification purposes a test and reference polypeptidelength of 100 amino acids are compared. No more than 10% (i.e., 10 outof 100) amino acids in the test polypeptide differ from that of thereference polypeptides. Similar comparisons can be made between a testand reference polynucleotides. Such differences can be represented aspoint mutations randomly distributed over the entire length of an aminoacid sequence or they can be clustered in one or more locations ofvarying length up to the maximum allowable, e.g., 10/100 amino aciddifference (approximately 90% identity). Differences are defined asnucleic acid or amino acid substitutions, or deletions.

As used herein, the phrase “sequence-related proteins” refers toproteins that have at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95% amino acid identity or homology witheach other.

As used herein, families of non-related proteins or“sequence-non-related proteins” refers to proteins that have less than50%, less than 40%, less than 0%, less than 20% amino acid identity orhomology with each other.

As used herein, it also is understood that the terms “substantiallyidentical” or “similar” varies with the context as understood by thoseskilled in the relevant art.

As used herein, heterologous or foreign nucleic acid, such as DNA andRNA, are used interchangeably and refer to DNA or RNA that does notoccur naturally as part of the genome in which it is present or which isfound in a location or locations in the genome that differ from that inwhich it occurs in nature. Heterologous nucleic acid is generally notendogenous to the cell into which it is introduced, but has beenobtained from another cell or prepared synthetically. Generally,although not necessarily, such nucleic acid encodes RNA and proteinsthat are not normally produced by the cell in which it is expressed.Heterologous DNA herein encompasses any DNA or RNA that one of skill inthe art would recognize or consider as heterologous or foreign to thecell in which it is expressed. Heterologous DNA and RNA can also encodeRNA or proteins that mediate or alter expression of endogenous DNA byaffecting transcription, translation, or other regulatable biochemicalprocesses. Examples of heterologous nucleic acid include, but are notlimited to, nucleic acid that encodes traceable marker proteins, such asa protein that confers drug resistance, nucleic acid that encodestherapeutically effective substances, such as anti-cancer agents,enzymes and hormones, and DNA that encodes other types of proteins, suchas antibodies.

Hence, herein heterologous DNA or foreign DNA, includes a DNA moleculenot present in the exact orientation and position as the counterpart DNAmolecule found in the genome. It can also refer to a DNA molecule fromanother organism or species (i.e., exogenous).

As used herein, a therapeutically effective dose refers to that amountof the compound sufficient to result in amelioration of symptoms ofdisease.

As used herein, isolated with reference to a nucleic acid molecule orpolypeptide or other biomolecule means that the nucleic acid orpolypeptide has separated from the genetic environment from which thepolypeptide or nucleic acid were obtained. It can also mean altered fromthe natural state. For example, a polynucleotide or a polypeptidenaturally present in a living animal is not “isolated,” but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated,” as the term is employed herein. Thus, apolypeptide or polynucleotide produced and/or contained within arecombinant host cell is considered isolated. Also intended as an“isolated polypeptide” or an “isolated polynucleotide” are polypeptidesor polynucleotides that have been purified, partially or substantially,from a recombinant host cell or from a native source. For example, arecombinantly produced version of a compound can be substantiallypurified by the one-step method described in Smith et al., Gene,67:31-40, 1988. The terms isolated and purified are sometimes usedinterchangeably.

Thus, by “isolated” is meant that the nucleic is free of the codingsequences of those genes that, in the naturally-occurring genome of theorganism (if any) immediately flank the gene encoding the nucleic acidof interest. Isolated DNA can be single-stranded or double-stranded, andcan be genomic DNA, cDNA, recombinant hybrid DNA, or synthetic DNA. Itcan be identical to a starting DNA sequence, or can differ from suchsequence by the deletion, addition, or substitution of one or morenucleotides.

Isolated or purified as it refers to preparations made from biologicalcells or hosts means any cell extract containing the indicated DNA orprotein including a crude extract of the DNA or protein of interest. Forexample, in the case of a protein, a purified preparation can beobtained following an individual technique or a series of preparative orbiochemical techniques and the DNA or protein of interest can be presentat various degrees of purity in these preparations. The procedures caninclude for example, but are not limited to, ammonium sulfatefractionation, gel filtration, ion exchange change chromatography,affinity chromatography, density gradient centrifugation andelectrophoresis.

A preparation of DNA or protein that is “substantially pure” or“isolated” should be understood to mean a preparation free fromnaturally occurring materials with which such DNA or protein is normallyassociated in nature. “Essentially pure” should be understood to mean a“highly” purified preparation that contains at least 95% of the DNA orprotein of interest.

A cell extract that contains the DNA or protein of interest should beunderstood to mean a homogenate preparation or cell-free preparationobtained from cells that express the protein or contain the DNA ofinterest. The term “cell extract” is intended to include culture media,especially spent culture media from which the cells have been removed.

As used herein, “a targeting agent” refers to any molecule that can bindanother target-molecule, such as an antibody, receptor, or ligand.

As used herein, receptor refers to a biologically active molecule thatspecifically binds to (or with) other molecules. The term “receptorprotein” can be used to more specifically indicate the proteinaceousnature of a specific receptor.

As used herein, recombinant refers to any progeny formed as the resultof genetic engineering.

As used herein, a promoter region refers to the portion of DNA of a genethat controls transcription of the DNA to which it is operativelylinked. The promoter region includes specific sequences of DNA that aresufficient for RNA polymerase recognition, binding and transcriptioninitiation. This portion of the promoter region is referred to as thepromoter. In addition, the promoter region includes sequences thatmodulate this recognition, binding and transcription initiation activityof the RNA polymerase. These sequences can be cis acting or can beresponsive to trans acting factors. Promoters, depending upon the natureof the regulation, can be constitutive or regulated.

As used herein, the phrase “operatively linked” generally means thesequences or segments have been covalently joined into one piece of DNA,whether in single or double stranded form, whereby control or regulatorysequences on one segment control or permit expression or replication orother such control of other segments. The two segments are notnecessarily contiguous. For gene expression a DNA sequence and aregulatory sequence(s) are connected in such a way to control or permitgene expression when the appropriate molecular, e.g., transcriptionalactivator proteins, are bound to the regulatory sequence(s).

As used herein, production by recombinant means by using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA, including cloning expressionof genes and methods, such as gene shuffling and phage display withscreening for desired specificities.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic DNA thatresults in more than one type of mRNA.

As used herein, a composition refers to any mixture of two or moreproducts or compounds. It can be a solution, a suspension, liquid,powder, a paste, aqueous, non-aqueous or any combination thereof.

As used herein, a combination refers to any association between two ormore items.

As used herein, substantially identical to a product means sufficientlysimilar so that the property of interest is sufficiently unchanged sothat the substantially identical product can be used in place of theproduct.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of exemplary vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Exemplary vectors are thosecapable of autonomous replication and/or expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors.” In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. “Plasmid” and “vector” areused interchangeably as the plasmid is the most commonly used form ofvector. Other such other forms of expression vectors that serveequivalent functions and that become known in the art subsequentlyhereto.

As used herein, vector also is used interchangeable with “virus vector”or “viral vector. In this case, which will be clear from the context,the “vector” is not self-replicating. Viral vectors are engineeredviruses that are operatively linked to exogenous genes to transfer (asvehicles or shuttles) the exogenous genes into cells.

As used herein, transduction refers to the process of gene transfer intoand expression in mammalian and other cells mediated by viruses.Transfection refers to the process when mediated by plasmids.

As used herein, transformation refers to the process of gene transferinto and expression in bacterial cells mediated by plasmids.

As used herein, “allele,” which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for the gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide, or several nucleotides, and caninclude substitutions, deletions, and insertions of nucleotides. Anallele of a gene also can be a form of a gene containing a mutation.

As used herein, the term “gene” or “recombinant gene” refers to anucleic acid molecule comprising an open reading frame and including atleast one exon and (optionally) an intron sequence. A gene can be eitherRNA or DNA. Genes can include regions preceding and following the codingregion (leader and trailer).

As used herein, “intron” refers to a DNA sequence present in a givengene which is spliced out during mRNA maturation.

As used herein, “nucleotide sequence complementary to the nucleotidesequence set forth in SEQ ID NO:” refers to the nucleotide sequence ofthe complementary strand of a nucleic acid strand having the particularSEQ ID NO:. The term “complementary strand” is used hereininterchangeably with the term “complement.” The complement of a nucleicacid strand can be the complement of a coding strand or the complementof a non-coding strand. When referring to double stranded nucleic acids,the complement of a nucleic acid having a particular SEQ ID NO: refersto the complementary strand of the strand set forth in the particularSEQ ID NO: or to any nucleic acid having the nucleotide sequence of thecomplementary strand of the particular SEQ ID NO:. When referring to asingle stranded nucleic acid having a nucleotide sequence correspondingto a particular SEQ ID NO:, the complement of this nucleic acid is anucleic acid having a nucleotide sequence which is complementary to thatof the particular SEQ ID NO:.

As used herein, the term “coding sequence” refers to that portion of agene that encodes an amino acid sequence of a protein.

As used herein, the term “sense strand” refers to that strand of adouble-stranded nucleic acid molecule that has the sequence of the mRNAthat encodes the amino acid sequence encoded by the double-strandednucleic acid molecule.

As used herein, the term “antisense strand” refers to that strand of adouble-stranded nucleic acid molecule that is the complement of thesequence of the mRNA that encodes the amino acid sequence encoded by thedouble-stranded nucleic acid molecule.

As used herein, an “array” refers to a collection of elements, such asnucleic acid molecules, containing three or more members. An addressablearray is one in which the members of the array are identifiable,typically by position on a solid phase support or by virtue of anidentifiable or detectable label, such as by color, fluorescence,electronic signal (i.e., RF, microwave or other frequency that does notsubstantially alter the interaction of the molecules of interest), barcode or other symbology, chemical or other such label. In certainembodiments, the members of the array are immobilized to discreteidentifiable loci on the surface of a solid phase or directly orindirectly linked to or otherwise associated with the identifiablelabel, such as affixed to a microsphere or other particulate support(herein referred to as beads) and suspended in solution or spread out ona surface.

As used herein, a “support” (also referred to as a matrix support, amatrix, an insoluble support or solid support) refers to any solid orsemisolid or insoluble support to which a molecule of interest,typically a biological molecule, organic molecule or biospecific ligandis linked or contacted. Such materials include any materials that areused as affinity matrices or supports for chemical and biologicalmolecule syntheses and analyses, such as, but are not limited to:polystyrene, polycarbonate, polypropylene, nylon, glass, dextran,chitin, sand, pumice, agarose, polysaccharides, dendrimers, buckyballs,polyacryl-amide, silicon, rubber, and other materials used as supportsfor solid phase syntheses, affinity separations and purifications,hybridization reactions, immunoassays and other such applications. Thematrix herein can be particulate or can be in the form of a continuoussurface, such as a microtiter dish or well, a glass slide, a siliconchip, a nitrocellulose sheet, nylon mesh, or other such materials. Whenparticulate, typically the particles have at least one dimension in the5-10 mm range or smaller. Such particles, referred collectively hereinas “beads,” are often, but not necessarily, spherical. Such reference,however, does not constrain the geometry of the matrix, which can be anyshape, including random shapes, needles, fibers, and elongated. Roughlyspherical “beads,” particularly microspheres that can be used in theliquid phase, also are contemplated. The “beads” can include additionalcomponents, such as magnetic or paramagnetic particles (see, e.g.,Dynabeads (Dynal, Oslo, Norway)) for separation using magnets, as longas the additional components do not interfere with the methods andanalyses herein.

As used herein, a “matrix” or “support particles” refers to matrixmaterials that are in the form of discrete particles. The particles haveany shape and dimensions, but typically have at least one dimension thatis 100 mm or less, 50 mm or less, 10 mm or less, 1 mm or less, 100 μm orless, 50 μm or less and typically have a size that is 100 mm³ or less,50 mm³ or less, 10 mm³ or less, and 1 mm³ or less, 100 μm³ or less andcan be order of cubic microns. Such particles are collectively called“beads.”

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, Biochem., 11:942-944(1972)).

B. Directed Evolution

To date, there have been three general approaches described for proteindirected evolution based on mutagenesis.

1. Pure Random Mutagenesis

Random mutagenesis methodology requires that the amino acids in thestarting protein sequence are replaced by all (or a group) of the 20amino acids. Either single or multiple replacements at different aminoacid positions are generated on the same molecule, at the same time. Therandom mutagenesis method relies on a direct search for fitnessimprovement based on random amino acid replacement and sequence changesat multiple amino acid positions. In this approach neither the aminoacid position (first dimension) nor the amino acid type (seconddimension) are restricted; and everything possible is generated andtested. Multiple replacements can randomly happen at the same time onthe same molecule. For example, random mutagenesis methods are widelyused to develop antibodies with higher affinity for its ligand, by thegeneration of random-sequence libraries of antibody molecules, followedby expression and screening using filamentous phages.

2. Restricted Random Mutagenesis

Restricted random mutagenesis methods introduce either all of the 20amino acids or DNA-biased residues. The bias is based on the sequence ofthe DNA and not on that of the protein, in a stochastic orsemi-stochastic manner, respectively, within restricted or predefinedregions of the protein, known in advance to be involved in thebiological activity being “evolved.” This method relies on a directsearch for fitness improvement based on random amino acid replacementand sequence changes at either restricted or multiple amino acidpositions. In this approach the scanning can be restricted to selectedamino acid positions and/or amino acid types, while material changescontinue to be random in position and type. For example, the amino acidposition can be restricted by prior selection of the target region to bemutated (selection of target region is based upon prior knowledge onprotein structure/function); while the amino acid type is not primarilyrestricted as replacing amino acids are stochastically or at most“semi-stochastically” chosen. As an example, this method is used tooptimize known binding sites on proteins, including hormone-receptorsystems and antibody-epitope systems.

3. Non-Restricted Rational Mutagenesis

Rational mutagenesis is a two-step process and is described inco-pending U.S. application Ser. No. 10/022,249. Briefly, the first steprequires amino acid scanning where all and each of the amino acids inthe starting protein sequence are replaced by a third amino acid ofreference (e.g., alanine). Only a single amino acid is replaced on eachprotein molecule at a time. A collection of protein molecules having asingle amino acid replacement is generated such that molecules differfrom each other by the amino acid position at which the replacement hastaken place. Mutant DNA molecules are designed, generated by mutagenesisand cloned individually, such as in addressable arrays, such that theyare physically separated from each other and such that each one is thesingle product of an independent mutagenesis reaction. Mutant proteinmolecules derived from the collection of mutant nucleic acid moleculesalso are physically separated from each other, such as by formatting inaddressable arrays. Activity assessment on each protein molecule allowsfor the identification of those amino acid positions that result in adrop in activity when replaced, thus indicating the involvement of thatparticular amino acid position in the protein's biological activityand/or conformation that leads to fitness of the particular featurebeing evolved. Those amino acid positions are referred to as HITs. Atthe second step, a new collection of molecules is generated such thateach molecule differs from each of the others by the amino acid presentat the individual HIT positions identified in step 1. All 20 amino acids(19 remaining) are introduced at each of the HIT positions identified instep 1; while each individual molecule contains, in principle, one andonly one amino acid replacement. Mutant DNA molecules are designed,generated by mutagenesis and cloned individually, such as in addressablearrays, such that they are physically separated from each other and suchthat each one is the single product of an independent mutagenesisreaction. Mutant protein molecules derived from the collection of mutantDNA molecules also are physically separated from each other, such as byformatting in addressable arrays. Activity assessment then isindividually performed on each individual mutant molecule. The newlygenerated mutants that lead to a desired alteration (such as animprovement) in a protein activity are referred to as LEADs. This methodpermits an indirect search for activity alteration, such as improvement,based on one rational amino acid replacement and sequence change at asingle amino acid position at a time, in search of a new, unpredictedamino acid sequence at some unpredicted regions along a protein toproduce a protein that exhibits a desired activity or altered activity,such as better performance than the starting protein.

In this approach, neither the amino acid position nor the replacingamino acid type are restricted. Full length protein scanning isperformed during the first step to identify HIT positions, and then all20 amino acids are tested at each of the HIT positions, to identify LEADsequences; while, as a starting point, only one amino acid at a time isreplaced on each molecule. The selection of the target region (HITs andsurrounding amino acids) for the second step is based upon experimentaldata on activity obtained in the first step. Thus, no prior knowledge ofprotein structure and/or function is necessary. Using this approach,LEAD sequences have been found on proteins that are located at regionsof the protein not previously known to be involved in the particularbiological activity being optimized; thus emphasizing the power of thisapproach to discover unpredictable regions (HITs) as targets for fitnessimprovement.

C. 2-Dimensional Rational Scanning (2D Scanning)

The 2-Dimensional rational scanning (or “2-dimensional scanning”)methods for protein rational evolution provided herein (see, alsocopending U.S. application Ser. No. 10/658,355, filed Sep. 8, 2003,based on U.S. provisional application Ser. Nos. 60/457,063 and60/410,258) are based on scanning over two dimensions. The firstdimension scanned is amino acid position along the protein sequence toidentify is-HIT target positions, and the second dimension is the aminoacid type selected for replacing a particular is-HIT amino acidposition. An advantage of the 2-dimensional scanning methods providedherein is that at least one, and typically both, of the amino acidposition scan and/or the replacing amino acid scan can be restrictedsuch that fewer than all amino acids on the protein-backbone areselected for amino acid replacement; and/or fewer than all of theremaining 19 amino acids available to replace an original, such asnative, amino acid are selected for replacement.

In particular embodiments, based on i) the particular protein propertiesto be evolved, ii) the protein's amino acid sequence, and iii) the knownproperties of the individual amino acids, a number of target positionsalong the protein sequence are selected, in silico, as “is-HIT targetpositions.” This number of is-HIT target positions is as large aspossible such that all reasonably possible target positions for theparticular feature being evolved are included. In particular,embodiments where a restricted number of is-HIT target positions areselected for replacement, the amino acids selected to replace the is-HITtarget positions on the particular protein being optimized can be eitherall of the remaining 19 amino acids or, more frequently, a morerestricted group comprising selected amino acids that are contemplatedto have the desired effect on protein activity. In another embodiment,so long as a restricted number of replacing amino acids are used, all ofthe amino acid positions along the protein backbone can be selected asis-HIT target positions for amino acid replacement. Mutagenesis then isperformed by the replacement of single amino acid residues at specificis-HIT target positions on the protein backbone (e.g., “one-by-one,”such as in addressable arrays), such that each individual mutantgenerated is the single product of each single mutagenesis reaction.Mutant DNA molecules are designed, generated by mutagenesis and clonedindividually, such as in addressable arrays, such that they arephysically separated from each other and that each one is the singleproduct of an independent mutagenesis reaction. Mutant protein moleculesderived from the collection of mutant DNA molecules also are physicallyseparated from each other, such as by formatting in addressable arrays.Thus, a plurality of mutant protein molecules are produced. Each mutantprotein contains a single amino acid replacement at only one of theis-HIT target positions. Activity assessment is then individuallyperformed on each individual protein mutant molecule, following proteinexpression and measurement of the appropriate activity. An example ofpractice of this method is shown in the Example in which mutant IFNαmolecules and IFNβ molecules are produced.

The newly generated proteins that lead to altered, typicallyimprovement, in a target protein activity are referred to as LEADs. Thismethod relies on an indirect search for protein improvement for aparticular activity, such as increased resistance to proteolysis, basedon a rational amino acid replacement and sequence change at single or,in another embodiment, a limited number of amino acid positions at atime. As a result, optimized proteins that have new amino acid sequencesat some regions along the protein that perform better (at a particulartarget activity or other property) than the starting protein areidentified and isolated.

1. Identifying In-Silico HITs

Provided herein is a method for directed evolution that includesidentifying and selecting (using in silico analysis) specific aminoacids and amino acid positions (referred to herein as is-HITs) along theprotein sequence that are contemplated to be directly or indirectlyinvolved in the feature being evolved. As noted, the 2-dimensionalscanning methods provided include the following two-steps. The firststep is an in silico search of a target protein's amino acid sequence toidentify all possible amino acid positions that potentially can betargets for the activity being evolved. This is effected, for example,by assessing the effect of amino acid residues on the property(ies) tobe altered on the protein, using any known standard software. Theparticulars of the in silico analysis is a function of the property tobe modified. For example, in the example herein, a property that isaltered resistance of the protein to proteolysis. To determine aminoacid residues that are potential targets as is-HITs, in this example,all possible target residues for proteases were first identified. The3-dimensional structure of the protein was then considered in order toidentify surface residues. Comparison of exposed residues withproteolytically cleavable residues yields residues that are targets forchange.

Once identified, these amino acid positions or target sequences arereferred to as “is-HITs” (in silico HITs). In silico HITs are defined asthose amino acid positions (or target positions) that potentially areinvolved in the “evolving” feature, such as increased resistance toproteolysis. In one embodiment, the discrimination of the is-HITs amongall the amino acid positions in a protein sequence is made based on i)the amino acid type at each position in addition to, whenever availablebut not necessarily, ii) the information on the protein secondary ortertiary structure. In silico HITs constitute a collection of mutantmolecules such that all possible amino acids, amino acid positions ortarget sequences potentially involved in the evolving feature arerepresented. No strong theoretical discrimination among amino acids oramino acid positions is made at this stage.

In silico HIT positions are spread over the full length of the proteinsequence. In one embodiment, only a single is-HIT amino acid at a timeis replaced on the target protein. In another embodiment, a limitednumber of is-HIT amino acids are replaced at the same time on the sametarget protein molecule. The selection of target regions (is-HITs andsurrounding amino acids) for the second step is based upon rationalassumptions and predictions. No prior knowledge of proteinstructure/function is necessary. Hence, the 2-dimensional scanningmethodology provided herein does not require any previous knowledge ofthe 3-dimensional conformational structure of the protein.

Any protein known or otherwise available to those of skill in the art issuitable for modification using the directed evolution methods providedherein, including cytokines (e.g., IFNα-2b) or any other proteins thathave previously been mutated or optimized.

A variety of parameters can be analyzed to determine whether or not aparticular amino acid on a protein might be involved in the evolvingfeature. For example, the information provided by crystal structures ofproteins can be rationally exploited in order to perform acomputer-assisted (in silico) analysis towards the prediction ofvariants with desired features. In a particular embodiment, a limitednumber of initial premises (typically no more than 2) are used todetermine the in silico HITs. In other embodiments, the number ofpremises used to determine the in silico hits can range from 1 to 10premises, including no more than 9, no more than 8, no more than 7, nomore than 6, no more than 5, no more than 4, no more than 3, but aretypically no more than 2 premises. It is important to the methodsprovided herein that the number of initial premises be kept to aminimum, so as to maintain the number of potential is-HITs at a maximum(here is where the methods provided are not limited by too muchprediction based on theoretical assumptions). When two premises areemployed, the first condition is typically the amino acid type itself,which is directly linked to the nature of the evolving feature. Forexample, if the goal were to change the optimum pH for an enzyme, thenthe replacing amino acids selected at this step for the replacement ofthe original sequence would be only those with a certain pKa value. Thesecond premise is typically related to the specific position of thoseamino acids along the protein structure. For example, some amino acidsmight be discarded if they are not expected to be exposed enough to thesolvent, even when they might have appropriate pKa values.

During the first step of identification of is-HITs according to themethods provided herein, each individual amino acid along the proteinsequence is considered individually to assess whether it is a candidatefor is-HIT. This search is done one-by-one and the decision on whetherthe amino acid is considered to be a candidate for a is-HIT is based on(1) the amino acid type itself; (2) the position on the amino acidsequence and protein structure if known; and (3) the predictedinteraction between that amino acid and its neighbors in sequence andspace.

Using the 3D-scanning methods provided herein, once one protein within afamily of proteins (e.g., IFNα-2b within the cytokine family) isoptimized using the methods provided herein for generating LEAD mutants,is-HITs can be identified on other or all proteins within a particularfamily by identifying the corresponding amino acid positions thereinusing structural homology analysis (based upon comparisons of the 3-Dstructures of the family members with original protein to identifycorresponding residues for replacement) as described hereinafter. Theis-HITs on family identified in this manner then can be subjected to thenext step of identifying replacing amino acids and further assayed toobtain LEADs or super-LEADs as described herein.

2. Identifying Replacing Amino Acids

Once the is-HITs target positions are selected, the next step isidentifying those amino acids that will replace the original, such asnative, amino acid at each is-HIT position to alter the activity levelfor the particular feature being evolved. The set of replacing aminoacids to be used to replace the original, such as native, amino acid ateach is-HIT position can be different and specific for the particularis-HIT position. The choice of the replacing amino acids takes intoaccount the need to preserve the physicochemical properties such ashydrophobicity, charge and polarity, of essential (e.g., catalytic,binding, etc.) residues. The number of replacing amino acids, of theremaining 19 non-native (or non-original) amino acids, that can be usedto replace a particular is-HIT target position ranges from 1 up to about19, from 1 up to about 15, from 1 up to about 10, from 1 up to about 9,from 1 up to about 8, from 1 up to about 7, from 1 up to about 6, from 1up to about 5, from 1 up to about 4, from 1 up to about 3, or from 1 to2 amino acid replacements.

Numerous methods of selecting replacing amino acids (also referred toherein as “replacement amino acids”) are well known in the art. Proteinchemists determined that certain amino acid substitutions commonly occurin related proteins from different species. As the protein stillfunctions with these substitutions, the substituted amino acids arecompatible with protein structure and function. Often, thesesubstitutions are to a chemically similar amino acid, but other types ofchanges, although relatively rare, can also occur.

Knowing the types of changes that are most and least common in a largenumber of proteins can assist with predicting alignments and amino acidsubstitutions for any set of protein sequences. Amino acid substitutionmatrices are used for this purpose.

In amino acid substitution matrices, amino acids are listed across thetop of a matrix and down the side, and each matrix position is filledwith a score that reflects how often one amino acid would have beenpaired with the other in an alignment of related protein sequences. Theprobability of changing amino acid A into amino acid B is assumed to beidentical to the reverse probability of changing B into A. Thisassumption is made because, for any two sequences, the ancestor aminoacid in the phylogenetic tree is usually not known. Additionally, thelikelihood of replacement should depend on the product of the frequencyof occurrence of the two amino acids and on their chemical and physicalsimilarities. A prediction of this model is that amino acid frequencieswill not change over evolutionary time (Dayhoff et al., Atlas of ProteinSequence and Structure, 5(3):345-352, 1978). Below are several exemplaryamino acid substitution matrices, including, but not limited to blocksubstitution matrix (BLOSUM), Jones, Gonnet, Fitch, Feng, McLachlan,Grantham, Miyata, Rao, Risler, Johnson and percent accepted mutation(PAM). Any such method known to those of skill in the art can beemployed.

a. Percent Accepted Mutation (PAM)

Dayhoff and coworkers developed a model of protein evolution thatresulted in the development of a set of widely used replacement matrices(Dayhoff et al., Atlas of Protein Sequence and Structure, 5(3):345-352,1978) termed percent accepted mutation matrices (PAM). In deriving thesematrices, each change in the current amino acid at a particular site isassumed to be independent of previous mutational events at that site.Thus, the probability of change of any amino acid A to amino acid B isthe same, regardless of the previous changes at that site and alsoregardless of the position of amino acid A in a protein sequence.

In the Dayhoff approach, replacement rates are derived from alignmentsof protein sequences that are at least 85% identical; this constraintensures that the likelihood of a particular mutation being the result ofa set of successive mutations is low. Because these changes are observedin closely related proteins, they represent amino acid substitutionsthat do not significantly change the function of the protein. Hence,they are called “accepted mutations,” as defined as amino acid changesthat are accepted by natural selection.

i. PAM Analysis

In particular embodiments of the methods provided herein, “PercentAccepted Mutation” (PAM; Dayhoff et al., Atlas of Protein Sequence andStructure, 5(3):345-352, 1978 FIG. 2) PAM values are used to select anappropriate group of replacement amino acids. PAM matrices wereoriginally developed to produce alignments between protein sequencesbased evolutionary distances. Because, in a family of proteins orhomologous (related) sequences, identical or similar amino acids (85%similarity) are shared, conservative substitutions for, or allowed pointmutations of the corresponding amino acid residues can be determinedthroughout an aligned reference sequence. Conservative substitutions ofa residue in a reference sequence are those substitutions that arephysically and functionally similar to the corresponding referenceresidues, e.g., that have a similar size, shape, electric charge,chemical properties, including the ability to form bonds such ascovalent and hydrogen bonds. Particularly suitable conservative aminoacid substitutions are those that show the highest scores and fulfillthe PAM matrix criteria in the form of “accepted point mutations.” Forexample, by comparing a family of scoring matrices, Dayhoff et al.,Atlas of Protein Sequence and Structure, 5(3):345-352, 1978, found aconsistently higher score significance when using PAM250 matrix toanalyze a variety of proteins, known to be distantly related.

ii. PAM 250

In a particular embodiment, the PAM250 matrix set forth in FIG. 2 isused for determining the replacing amino acids based on similaritycriteria. The PAM250 matrix uses data obtained directly from naturalevolution to facilitate the selection of replacing amino acids for theis-HITs to generate conservative mutations without much affecting theoverall protein function. By using the PAM250 matrix, candidatereplacing amino acids are identified from related proteins fromdifferent organisms.

b. Jones et al. and Gonnet et al.

This method (see, e.g., Jones et al., Comput. Appl. Biosci., 8:275-282,1992 and Gonnet et al., Science, 256:1433-1445, 1992) uses much of thesame methodology as Dayhoff (see below), but with modern databases. Thematrix of Jones et al., is extracted from Release 15.0 of the SWISS-PROTprotein sequence database. Point mutations totaling 59,160 from 16,130protein sequences were used to calculate a PAM250 (see below) matrix.

The matrix published by Gonnet et al., Science, 256:1433-1445, 1992, wasbuilt from a sequence database of 8,344,353 amino acid residues. Eachsequence was compared against the entire database, such that 1.7×10⁶subsequent matches resulted for the significant alignments. Thesematches were then used to generate a matrix with a PAM distance of 250.

c. Fitch and Feng et al.

Fitch, J. Mol. Evol., 16(1): 9-16, 1966, used an exchange matrix thatcontained for each pair (A, B) of amino acid types the minimum number ofnucleotides that must be changed to encode amino acid A instead of aminoacid B. Feng et al., J. Mol. Evol., 21: 112-125, 1985, used an enhancedversion of Fitch, J. Mol. Evol., 16(1): 9-16, 1966, to build aStructure-Genetic matrix. In addition to considering the minimum numberof base changes required to encode amino acid B instead of A, thismethod also considers the structural similarity of the amino acids.

d. McLachlan, Grantham and Miyata

McLachlan, J. Mol. Biol., 61:409-424 1971, used 16 protein families,each with 2 to 14 members. The 89 sequences were aligned and thepairwise exchange frequency, observed in 9280 substitutions, was used togenerate an exchange matrix with values varying from 0 to 9.

Grantham, Science, 185:862-864, 1974, considers composition, polarityand molecular volume of amino acid side-chains, properties that werehighly correlated to the relative substitution frequencies tabulated byMcLachlan, J. Mol. Biol., 61:409-424, 1971, to build the matrix.

Miyata, J. Mol. Evol., 12:219-236, 1979, uses the volume and polarityvalues of amino acids published by Grantham, Science, 185:862-864, 1974.For every amino acid type pair, the difference for both properties wascalculated and divided by the standard deviation of all the differences.The square root of the sum of both values is then used in the matrix.

e. Rao

Rao, J. Pept. Protein Res., 29:276-281, 1987, employs five amino acidproperties to create a matrix; namely, alpha-helical, beta-strand andreverse-turn propensities as well as polarity and hydrophobicity. Thestandardized properties were summed and the matrix rescaled to the sameaverage as that for PAM (Dayhoff et al., Atlas of Protein Sequence andStructure, 5(3):345-352, 1978).

f. Risler et al.

Risler et al., J. Mol. Biol., 204:1019-1029, 1988, aligned 32three-dimensional structures from 11 protein families by rigid-bodysuperposition of the backbone topology. Only substitutions wereconsidered where at least three adjacent and equivalent main-chain Cαatom pairs in the compared structures were each not more than 1.2 Åapart. A total of 2860 substitutions were considered and used to build amatrix based on χ² distance calculations.

g. Johnson et al.

Johnson et al., J. Mol. Biol., 233:716-738, 1993, derived their matrixfrom the tertiary structural alignment of 65 families in a database of235 structures created with the method of Sali et al., J. Mol. Biol.,212:403-428, 1990. Their examination of the substitutions was based onthe expected and observed ratios of occurrences and the final matrixvalues were taken as log 10 of the ratios.

h. Block Substitution Matrix (BLOSUM)

One empirical approach (Henikoff et al., Proc. Natl. Acad. Sci. USA,89:10915-10919, 1992) uses local, ungapped alignments of distantlyrelated sequences to derive the blocks amino acid substitution matrix(BLOSUM) series of matrices. The matrix values are based on the observedamino acid substitutions in a larger set of about 2000 conserved aminoacid patterns, termed blocks. These blocks act as signatures of familiesof related proteins. Matrices of this series are identified by a numberafter the matrix (e.g., BLOSUM50), which refers to the minimumpercentage identity of the blocks of multiple aligned amino acids usedto construct the matrix. It is noteworthy that these matrices aredirectly calculated without extrapolations, and are analogous totransition probability matrices P(T) for different values of T,estimated without reference to any rate matrix Q.

The outcome of these two steps set forth above, which is performed insilico is that: (1) the amino acid positions that will be the target formutagenesis are identified; these positions are referred to as is-HITs;(2) the replacing amino acids for the original, such as native, aminoacids at the is-HITs are identified, to provide a collection ofcandidate LEAD mutant molecules that are expected to perform differentfrom the native one. These are assayed for a desired optimized (orimproved or altered) biological activity.

3. Physical Construction of Mutant Proteins and Biological Assays

Once is-HITs are selected as set forth above, replacing amino acids areintroduced. Mutant proteins typically are prepared using recombinant DNAmethods and assessed in appropriate biological assays for the particularbiological activity (feature) optimized (see, e.g., Example 1). Anexemplary method of preparing the mutant proteins is by mutagenesis ofthe original, such as native, gene using methods well known in the art.Mutant molecules are generated one-by-one, such as in addressablearrays, such that each individual mutant generated is the single productof each single and independent mutagenesis reaction. Individualmutagenesis reactions are conducted separately, such as in addressablearrays where they are physically separated from each other. Once apopulation of sets of nucleic acid molecules encoding the respectivemutant proteins is prepared, each is separately introduced one-by-oneinto appropriate cells for the production of the corresponding mutantproteins. This can also be performed, for example, in addressable arrayswhere each set of nucleic acid molecules encoding a respective mutantprotein is introduced into cells confined to a discrete location, suchas in a well of a multi-well microtiter plate. Each individual mutantprotein is individually phenotypically characterized and performance isquantitatively assessed using assays appropriate for the feature beingoptimized (i.e., feature being evolved). Again, this step can beperformed in addressable arrays. Those mutants displaying a desiredincreased or decreased performance compared to the original, such asnative molecules are identified and designated LEADs. From the beginningof the process of generating the mutant DNA molecules up through thereadout and analysis of the performance results, each candidate LEADmutant is generated, produced and analyzed individually, such as fromits own address in an addressable array. The process is amenable toautomation.

D. 2-Dimensional Scanning of Proteins for Increased Resistance toProteolysis

The methods of 2-dimensional scanning permit preparation of proteinsmodified for a selected trait, activity or other phenotype. Amongmodifications of interest for therapeutic proteins are those thatincrease protection against protease digestion while maintaining therequisite biological activity. Such changes are useful for producinglonger-lasting therapeutic proteins.

The delivery of stable peptide and protein drugs to patients is a majorchallenge for the pharmaceutical industry. These types of drugs in thehuman body are constantly eliminated or taken out of circulation bydifferent physiological processes including internalization, glomerularfiltration and proteolysis. The latter is often the limiting processaffecting the half-life of proteins used as therapeutic agents inper-oral administration and either intravenous or intramuscularinjections.

The 2-dimensional scanning process for protein evolution is used toeffectively improve protein resistance to proteases and thus increaseprotein half-life in vitro and, ultimately in vivo. As noted, themethods provided herein for designing and generating highly stable,longer lasting proteins, or proteins having a longer half-life include:i) identifying some or all possible target sites on the protein sequencethat are susceptible to digestion by one or more specific proteases(these sites are referred to herein as is-HITs); ii) identifyingappropriate replacing amino acids, specific for each is-HIT, such thatupon replacement of one or more of the original, such as native, aminoacids at that specific is-HIT, they can be expected to increase theis-HIT's resistance to digestion by protease while at the same time,maintaining or improving the requisite biological activity of theprotein (these proteins with replaced amino acids are the “candidateLEADs”); iii) systematically introducing the specific replacing aminoacids (candidate LEADs) at every specific is-HIT target position togenerate a collection containing the corresponding mutant candidate leadmolecules. Mutants are generated, produced and phenotypicallycharacterized one-by-one, such as in addressable arrays, such that eachmutant molecule contains initially an amino acid replacement at only oneis-HIT site.

In particular embodiments, such as in subsequent rounds, mutantmolecules also can be generated that contain one or more amino acids atone or more is-HIT sites that have been replaced by candidate LEAD aminoacids. Those mutant proteins carrying one or more mutations at one ormore is-HITs, and that display improved protease resistance are calledLEADs (one mutation at one is-HIT) and super-LEADs (mutations at morethan one is-HIT).

The first step of the process takes into consideration existingknowledge from different domains:

(1) About the galenic and the delivery environment (tissue, organ orcorporal fluid) of the particular therapeutic protein in order toestablish a list of proteases more likely to be found in thatenvironment. For example, a therapeutic protein in per-oral applicationis likely to encounter typical proteases of the luminal gastrointestinaltract. In contrast, if this protein were injected in the bloodcirculation, serum proteases would be implicated in the proteolysis.Based on the specific list of proteases involved, the complete list ofall amino acid sequences that potentially could be targeted by theproteases in the list is determined.

(2) Since protease mixtures in the body are quite complex incomposition, almost all the residues in any target protein potentiallyare targeted for proteolysis (FIG. 1A). Nevertheless, proteins formspecific tri-dimensional structures where residues are more or lessexposed to the environment and protease action. It can be assumed thatthose residues constituting the core of a protein are inaccessible toproteases, while those more “exposed” to the environment are bettertargets for proteases. The probability for every specific amino acid tobe “exposed” and then to be accessible to proteases can be taken intoaccount to reduce the number of is-HIT. Consequently, the methods hereinconsider the analysis with respect to solvent “exposure” or“accessibility” for each individual amino acid in the protein sequence.Solvent accessibility of residues can alternatively be estimated,regardless of any previous knowledge of specific protein structuraldata, by using an algorithm derived from empirical amino acidprobabilities of accessibility, which is expressed in the followingequation (Boger et al., Reports of the Sixth International Congress inImmunology, p. 250, 1986):

${A(i)} = {\left\lbrack {\overset{6}{\underset{j = 1}{\sqcap}}{{\_\delta}\__{i + 4 - j}}} \right\rbrack*{\lbrack 0.62\rbrack^{- 6}.}}$Briefly, these are fractional probabilities (δ_(i)) determined for anamino acid (i) found on the surface of a protein, which are based uponstructural data from a set of several proteins. It is thus possible tocalculate the solvent accessibility (A) of an amino acid (A(i)) atsequence position (i−2 to i+3, onto a sliding window of length equal to6) that is within an average surface accessible to solvent of >20 squareangstroms (Å²).

The protease accessible target amino acids along the protein sequence,i.e., the amino acids to be replaced, are thus identified and arereferred to herein as in silico HITs (is-HITs).

Amino acids at the is-HITs then are replaced by residues that render thesequence less vulnerable (by a factor, for example, of 1%, 10%, 20%,30%, 40%, 50%, . . . 100% depending upon the protein) or invulnerable(substantially no detectable digestion within a set time period) toprotease digestion, while at the same time maintain a biologicalactivity or activities of interest of the protein. The choice of thereplacing amino acids is complicated by (1) the broad target specificityof certain proteases and (2) the need to preserve the physicochemicalproperties such as hydrophobicity, charge and polarity, of essential(e.g., catalytic, binding and/or other activities depending upon theprotein) residues. For use in the methods herein, the “Percent AcceptedMutation” values (PAM values; see, Dayhoff et al., Atlas of ProteinSequence and Structure, 5(3):345-352, 1978), FIG. 2) can be used asdescribed herein. PAM values, originally developed to produce alignmentsbetween protein sequences, are available in the form of probabilitymatrices, which reflect an evolutionary distance. Since, in a family ofproteins or homologous (related) sequences, identical or similar aminoacids (85% similarity) are shared, conservative substitutions for, or“allowed point mutations” of the corresponding amino acid residues canbe determined throughout an aligned reference sequence. As noted,conservative substitutions of a residue in a reference sequence arethose substitutions that are physically and functionally similar to thecorresponding reference residues e.g., that have a similar size, shape,electric charge, chemical properties, including the ability to formbonds such as covalent and hydrogen bonds. For example, conservativesubstitutions can be those that exhibit the highest scores and fulfillthe PAM matrix criteria in the form of “accepted point mutations.”

By comparing a family of scoring matrices, Dayhoff et al., Atlas ofProtein Sequence and Structure, 5(3):345-352, 1978), found consistentlyhigher score significance when using PAM250 matrix to analyze a varietyof proteins, known to be distantly related. For methods herein, thePAM250 matrix was selected for use. The PAM250 matrix is used, bylearning directly from natural evolution, to find replacing amino acidsfor the is-HITs to generate conservative mutations without affecting theprotein function. By using PAM250, candidate replacing amino acids areidentified from related proteins from different organisms.

An exemplary class of proteins that can be optimized according to themethods provided herein are the cytokines. For example, 2D-scanningmethods provided herein can be used to modify the following cytokines toincrease their stability as assessed by an increased resistance toproteolysis resulting in an increased protein half-life in thebloodstream or any other desired biological activity of the selectedprotein. Exemplary cytokines, include, but are not limited to:interleukin-10 (IL-10; SEQ ID NO: 200), interferon beta (IFN-β; SEQ IDNO: 196), interferon alpha-2a (IFNα-2a; SEQ ID NO: 182), interferonalpha-2b (IFNα-2b; SEQ ID NO: 1), and interferon gamma (IFN-γ, SEQ IDNO: 199), granulocyte colony stimulating factor (G-CSF; SEQ ID NO: 210),leukemia inhibitory factor (LIF; SEQ ID NO: 213), growth hormone (hGH;SEQ ID NO: 216), ciliary neurotrophic factor (CNTF; SEQ ID NO: 212),leptin (SEQ ID NO: 211), oncostatin M (SEQ ID NO: 214), interleukin-6(IL-6; SEQ ID NO: 217), interleukin-12 (IL-12; SEQ ID NO: 215),erythropoietin (EPO; SEQ ID NO: 201), granulocyte-macrophage colonystimulating factor (GM-CSF; SEQ ID NO: 202), interleukin-2 (IL-2; SEQ IDNO: 204), interleukin-3 (IL-3; SEQ ID NO: 205), interleukin-4 (IL-4; SEQID NO: 207), interleukin-5 (IL-5; SEQ ID NO: 208), interleukin-13(IL-13; SEQ ID NO: 209), Flt3 ligand (SEQ ID NO: 203) and stem cellfactor (SCF; SEQ ID NO: 206).

Accordingly, provided herein are modified cytokines that exhibitincreased resistance to proteolysis compared to the unmodified cytokine.The modified cytokines can be selected from among a member of theinterferons/interleukin-10 protein family, a member of the long-chaincytokine family; and a member of the short-chain cytokine family. Inparticular embodiments, the modified cytokines provided herein areselected from among: interleukin-10 (IL-10), interferon beta (IFNβ),interferon alpha-2a (IFNα-2a), interferon alpha-2b (IFNα-2b), andinterferon gamma (IFN-γ), granulocyte colony stimulating factor (G-CSF),leukemia inhibitory factor (LIF), human growth hormone (hGH), ciliaryneurotrophic factor (CNTF), leptin, oncostatin M, interleukin-6 (IL-6)and interleukin-12 (IL-12), erythropoietin (EPO), granulocyte-macrophagecolony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-3(IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13(IL-13), Flt3 ligand and stem cell factor (SCF). In one embodiment, themodified cytokine is an interferon, including modified interferon α-2b(IFNα-2b).

E. Rational Evolution of IFNα-2b for Increased Resistance to Proteolysis

IFNα-2b is used for a variety of applications. Typically it is used fortreatment of type B and C chronic hepatitis. Additional indicationsinclude, but are not limited to, melanomas, herpes infections, Kaposisarcomas and some leukemia and lymphoma cases. Patients receivinginterferon are subject to frequent repeat applications of the drug.Since such frequent injections generate uncomfortable physiological aswell as undesirable psychological reactions in patients, increasing thehalf-life of interferons and thus decreasing the necessary frequency ofinterferon injections, would be extremely useful to the medicalcommunity. For example, after injection of native human IFNα-2binjection in mice, as a model system, its presence can be detected inthe serum between 3 and 10 hours with a half-life of only around 4hours. The IFNα-2b completely disappears to undetectable levels by 18-24hours after injection. Provided herein are mutant variants of theIFNα-2b protein that display altered properties including: (a) highlyimproved stability as assessed by resistance to proteases in vitro andby pharmacokinetics studies in mice; and (b) at least comparablebiological activity as assessed by antiviral and antiproliferativeaction compared to both the unmodified and wild type native IFNα-2bprotein and to at least one pegylated derivative of the wild type nativeIFNα. As a result, the IFNα-2b mutant proteins provided herein confer ahigher half-life and at least comparable antiviral and antiproliferationactivity (sufficient for a therapeutic effect) with respect to thenative sequence and to the pegylated derivatives molecules currentlybeing used for the clinical treatment of hepatitis C infection. SeeFIGS. 6(A)-6(N), 6(T) and 6(U). Thus, the optimized IFNα-2b proteinmutants that possess increased resistance to proteolysis and/orglomerular filtration provided herein result in a decrease in thefrequency of injections needed to maintain a sufficient drug level inserum, leading to i) higher comfort and acceptance by patients, ii)lower doses necessary to achieve comparable biological effects, and iii)as a consequence of (ii), an attenuation of the (dose-dependent)secondary effects observed in humans.

In particular embodiments, the half-life of the IFNα-2b and IFNα-2amutants provided herein is increased by an amount selected from at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 100%, at least150%, at least 200%, at least 250%, at least 300%, at least 350%, atleast 400%, at least 450%, at least 500% or more, when compared to thehalf-life of native human IFNα-2b and IFNα-2a in either human blood,human serum or an in vitro mixture containing one or more proteases. Inother embodiments, the half-life of the IFNα-2b and IFNα-2a mutantsprovided herein is increased by an amount selected from at least 6times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times,900 times, 1000 times, or more, when compared to the half-life of nativehuman IFNα-2b and IFNα-2a in either human blood, human serum or an invitro mixture containing one or more proteases.

Two methodologies were used herein to increase the stability of IFNα-2bby amino acid replacement: i) amino acid replacement that leads tohigher resistance to proteases by direct destruction of the proteasetarget residue or sequence, while either maintaining or improving therequisite biological activity (e.g., antiviral activity,antiproliferation activity), and/or ii) amino acid replacement thatleads to a different pattern of N-glycosylation, thus decreasing bothglomerular filtration and sensitivity to proteases, while eitherimproving or maintaining the requisite biological activity (e.g.,antiviral activity, antiproliferation activity).

The 2D-scanning methods provided herein were used to identify the aminoacid changes on IFNα-2b that lead to an increase in stability whenchallenged either with proteases, human blood lysate or human serum.Increasing protein stability to proteases, human blood lysate or humanserum, and/or increasing the molecular size is contemplated herein toprovide a longer in vivo half-life for the particular protein molecules,and thus to a reduction in the frequency of necessary injections intopatients. The biological activities that were measured for the IFNα-2bmolecules are i) their capacity to inhibit virus replication when addedto permissive cells previously infected with the appropriate virus, andii) their capacity to stimulate cell proliferation when added to theappropriate cells. Prior to the measurement of biological activity,IFNα-2b molecules were challenged with proteases, human blood lysate orhuman serum during different incubation times. The biological activitymeasured, corresponds then to the residual biological activity followingexposure to the protease-containing mixtures.

As set forth above, provided herein are methods for the development ofIFNα-2b and IFNα-2a molecules that, while maintaining the requisitebiological activity intact, have been rendered less susceptible todigestion by blood proteases and therefore display a longer half-life inblood circulation. In this particular example, the method used includedthe following specific steps as set forth in Example 2:

1) Identifying some or all possible target sites on the protein sequencethat are susceptible to digestion by one or more specific proteases(these sites are the is-HITs) and

2) Identifying appropriate replacing amino acids, specific for eachis-HIT, such that if used to replace one or more of the original aminoacids at that specific is-HIT, they can be expected to increase theis-HIT's resistance to digestion by protease while at the same time,keeping the biological activity of the protein unchanged (thesereplacing amino acids are the “candidate LEADs”).

As set forth in Example 2, the 3-dimensional structure of IFNα-2bobtained from the NMR structure of IFNα-2a (PDB code 1ITF) was used toselect only those residues exposed to solvent from a list of residuesalong the IFNα-2b and IFNα-2a sequence which can be recognized as asubstrate for different enzymes present in the serum. Residue 1corresponds to the first residue of the mature peptide IFNα-2b (SEQ IDNO:1) encoded by nucleotides 580-1074 of sequence accession No. J00207.Using this approach, the following 42 amino acid target positions wereidentified as is-HITs on IFNα-2b or IFNα-2a, which numbering is that ofthe mature protein (SEQ ID NO:1 or SEQ ID NO:182, respectively): L3, P4,R12, R13, M16, R22, K23 or R23, F27, L30, K31, R33, E41, K49, E58, K70,E78, K83, Y89, E96, E107, P109, L110, M111, E113, L117, R120, K121,R125, L128, K131, E132, K133, K134, Y135, P137, M148, R149, E159, L161,R162, K164, and E165. Each of these positions was replaced by residuesdefined as compatible by the substitution matrix PAM250 while at thesame time not generating any new substrates for proteases. For these 42is-HITs, the residue substitutions determined by PAM250 analysis were asfollows:

R to H, Q

E to H, Q

K to Q, T

L to V, I

M to I, V

P to A, S

Y to I, H.

1. Modified IFNα-2b Proteins with Single Amino Acid Substitutions(is-HITS)

Among the mutant proteins provided herein, are mutant IFNα-2b proteinsthat have increased resistance proteolysis compared to the unmodified,typically wild-type, protein. The mutant IFNα-2b proteins include thoseselected from among proteins containing more single amino acidreplacements in SEQ ID NO:1, corresponding to: L by V at position 3; Lby I at position 3; P by S at position 4; P by A at position 4; R by Hat position 12; R by Q at position 12; R by H at position 13; R by Q atposition 13; M by V at position 16; M by I at position 16; R by H atposition 22; R by Q at position 22; R by H at position 23; R by Q atposition 23; F by I at position 27; F by V at position 27; L by V atposition 30; L by I at position 30; K by Q at position 31; K by T atposition 31; R by H at position 33; R by Q at position 33; E by Q atposition 41; E by H at position 41; K by Q at position 49; K by T atposition 49; E by Q at position 58; E by H at position 58; K by Q atposition 70; K by T at position 70; E by Q at position 78; E by H atposition 78; K by Q at position 83; K by T at position 83; Y by H atposition 89; Y by I at position 89; E by Q at position 96; E by H atposition 96; E by Q at position 107; E by H at position 107; P by S atposition 109; P by A at position 109; L by V at position 110; L by I atposition 110; M by V at position 111; M by I at position 111; E by Q atposition 113; E by H at position 113; L by V at position 117; L by I atposition 117; R by H at position 120; R by Q at position 120; K by Q atposition 121; K by T at position 121; R by H at position 125; R by Q atposition 125; L by V at position 128; L by I at position 128; K by Q atposition 131; K by T at position 131; E by Q at position 132; E by H atposition 132; K by Q at position 133; K by T at position 133; K by Q atposition 134; K by T at position 134; Y by H at position 135; Y by I atposition 135; P by S at position 137; P by A at position 137; M by V atposition 148; M by I at position 148; R by H at position 149; R by Q atposition 149; E by Q at position 159; E by H at position 159; L by V atposition 161; L by I at position 161; R by H at position 162; R by Q atposition 162; K by Q at position 164; K by T at position 164; E by Q atposition 165; and E by H at position 165.

2. LEAD Identification

Next the specific replacing amino acids (candidate LEADs) aresystematically introduced at every specific is-HIT position to generatea collection containing the corresponding mutant IFNα-2b DNA molecules,as set forth in Example 2. The mutant DNA molecules were used to producethe corresponding mutant IFNα-2b protein molecules by transformation ortransfection into the appropriate cells. These protein mutants wereassayed for (i) protection against proteolysis, (ii) antiviral andantiproliferation activity in vitro, (iii) pharmacokinetics in mice. Ofparticular interest are mutations that increase these activities of theIFNα-2b mutant proteins compared to unmodified wild type IFNα-2b proteinand to pegylated derivates of the wild type protein. Based on theresults obtained from these assays, each individual IFNα-2b variant wasassigned a specific activity. Those variant proteins displaying thehighest stability and/or resistance to proteolysis were selected asLEADs. The candidate LEADs that possessed at least as much residualantiviral activity following protease treatment as the control, nativeIFNα-2b, before protease treatment were selected as LEADs. The resultsare set forth in Table 2 of Example 2.

Using this method, the following mutants selected as LEADs are providedherein and correspond to the group of proteins containing one or moresingle amino acid replacements in SEQ ID NO:1, corresponding to: F by Vat position 27; R by H at position 33; E by Q at position 41; E by H atposition 41; E by Q at position 58; E by H at position 58; E by Q atposition 78; E by H at position 78; Y by H at position 89; E by Q atposition 107; E by H at position 107; P by A at position 109; L by V atposition 110; M by V at position 111; E by Q at position 113; E by H atposition 113; L by V at position 117; L by I at position 117; K by Q atposition 121; K by T at position 121; R by H at position 125; R by Q atposition 125; K by Q at position 133; K by T at position 133; and E by Qat position 159; E by H at position 159. Among these are mutations thatcan have multiple effects. For example, among mutations describedherein, are mutations that result in an increase of the IFNα-2b activityas assessed by detecting the requisite biological activity.

Also provided are IFNα-2b proteins that contain a plurality of mutationsbased on the LEADs (see, e.g., Tables 6 and 7, EXAMPLE 5, which listscandidate LEADs and LEAD sites), are generated. These IFNα-2b proteinshave activity that is further optimized. Examples of such proteins aredescribed in the EXAMPLES. Other combinations of mutations can beprepared and tested as described herein to identify other LEADs ofinterest, particularly those that have further increased IFNα-2bantiviral activity or further increased resistance to proteolysis.

Also provided herein are modified IFNα-2b or IFNα-2a cytokines selectedfrom among proteins comprising one or more single amino acidreplacements in SEQ ID NOS:1 or 182, corresponding to the replacementof: N by D at position 45; D by G at position 94; G by R at position102; A by G at position 139; or any combination thereof. Theseparticular proteins have also been found herein to have increasedresistance to proteolysis.

In another embodiment, IFNα-2b and IFNα-2a proteins that contain aplurality of mutations based on the LEADs (see Tables in the EXAMPLES,listing the candidate LEADs and LEAD sites), are produced to produceIFNα-2b and IFNα-2a proteins that have activity that is furtheroptimized. Examples of such proteins are described herein. Othercombinations of mutations can be prepared and tested as described hereinto identify other LEADs of interest, particularly those that havefurther increased IFNα-2b and IFNα-2a antiviral activity or furtherincreased resistance to proteolysis.

3. N-glycosylation Site Addition

In additional embodiments, N-glycosylation sites can be added toincrease resistance to proteolysis while maintaining or improving therequisite biological activity. Exemplary N-glycosylation mutantscontaining duo-amino acid replacements corresponding to the N-X-S orN-X-T consensus sequences are set forth in Example 3. Accordingly,provided herein are IFNα-2b and IFNα-2a mutant proteins having anincreased resistance to proteolysis compared to unmodified IFNα-2b andIFNα-2a, selected from among proteins comprising one or more sets ofduo-amino acid replacements in SEQ ID NO:1, corresponding to:

-   D by N at position 2 and P by S at position 4;-   D by N at position 2 and P by T at position 4;-   L by N at position 3 and Q by S at position 5;-   L by N at position 3 and Q by T at position 5;-   P by N at position 4 and T by S at position 6;-   P by N at position 4 and T by T at position 6;-   Q by N at position 5 and H by S at position 7;-   Q by N at position 5 and H by T at position 7;-   T by N at position 6 and S by S at position 8;-   T by N at position 6 and S by T at position 8;-   H by N at position 7 and L by S at position 9;-   H by N at position 7 and L by T at position 9;-   S by N at position 8 and G by S at position 10;-   S by N at position 8 and G by T at position 10;-   L by N at position 9 and S by S at position 11;-   L by N at position 9 and S by T at position 11;-   M by N at position 21 and K by S at position 23;-   M by N at position 21 and K by T at position 23;-   R by N at position 22 and I by S at position 24;-   R by N at position 22 and I by T at position 24;-   K or R by N at position 23 and S by S at position 25;-   K or R by N at position 23 and S by T at position 25;-   I by N at position 24 and L by S at position 26;-   I by N at position 24 and L by T at position 26;-   S by N at position 25 and F by S at position 27;-   S by N at position 25 and F by T at position 27;-   L by N at position 26 and S by S at position 28;-   L by N at position 26 and S by T at position 28;-   S by N at position 28 and L by S at position 30;-   S by N at position 28 and L by T at position 30;-   L by N at position 30 and D by S at position 32;-   L by N at position 30 and D by T at position 32;-   K by N at position 31 and R by S at position 33;-   K by N at position 31 and R by T at position 33;-   D by N at position 32 and H by S at position 34;-   D by N at position 32 and H by T at position 34;-   R by N at position 33 and D by S at position 35;-   R by N at position 33 and D by T at position 35;-   H by N at position 34 and F by S at position 36;-   H by N at position 34 and F by T at position 36;-   D by N at position 35 and G by S at position 37;-   D by N at position 35 and G by T at position 37;-   F by N at position 36 and F by S at position 38;-   F by N at position 36 and F by T at position 38;-   G by N at position 37 and P by S at position 39;-   G by N at position 37 and P by T at position 39;-   F by N at position 38 and Q by S at position 40;-   F by N at position 38 and Q by T at position 40;-   P by N at position 39 and E by S at position 41;-   P by N at position 39 and E by T at position 41;-   Q by N at position 40 and E by S at position 42;-   Q by N at position 40 and E by T at position 42;-   E by N at position 41 and F by S at position 43;-   E by N at position 41 and F by T at position 43;-   E by N at position 42 and G by S at position 44;-   E by N at position 42 and G by T at position 44;-   F by N at position 43 and N by S at position 45;-   F by N at position 43 and N by T at position 45;-   G by N at position 44 and Q by S at position 46;-   G by N at position 44 and Q by T at position 46;-   N by N at position 45 and F by S at position 47;-   N by N at position 45 and F by T at position 47;-   Q by N at position 46 and Q by S at position 48;-   Q by N at position 46 and Q by T at position 48;-   F by N at position 47 and K by S at position 49;-   F by N at position 47 and K by T at position 49;-   Q by N at position 48 and A by S at position 50;-   Q by N at position 48 and A by T at position 50;-   K by N at position 49 and E by S at position 51;-   K by N at position 49 and E by T at position 51;-   A by N at position 50 and T by S at position 52;-   A by N at position 50 and T by T at position 52;-   S by N at position 68 and K by S at position 70;-   S by N at position 68 and K by T at position 70;-   K by N at position 70 and S by S at position 72;-   K by N at position 70 and S by T at position 72;-   A by N at position 75 and D by S at position 77;-   A by N at position 75 and D by T at position 77;-   D by N at position 77 and T by S at position 79;-   D by N at position 77 and T by T at position 79;-   I by N at position 100 and G by S at position 102;-   I by N at position 100 and G by T at position 102;-   Q by N at position 101 and V by S at position 103;-   Q by N at position 101 and V by T at position 103;-   G by N at position 102 and G by S at position 104;-   G by N at position 102 and G by T at position 104;-   V by N at position 103 and V by S at position 105;-   V by N at position 103 and V by T at position 105;-   G by N at position 104 and T by S at position 106;-   G by N at position 104 and T by T at position 106;-   V by N at position 105 and E by S at position 107;-   V by N at position 105 and E by T at position 107;-   T by N at position 106 and T by S at position 108;-   T by N at position 106 and T by T at position 108;-   E by N at position 107 and P by S at position 109;-   E by N at position 107 and P by T at position 109;-   T by N at position 108 and I by S at position 110;-   T by N at position 108 and I by T at position 110;-   K by N at position 134 and S by S at position 136;-   K by N at position 134 and S by T at position 136;-   S by N at position 154 and N by S at position 156;-   S by N at position 154 and N by T at position 156;-   T by N at position 155 and L by S at position 157;-   T by N at position 155 and L by T at position 157;-   N by N at position 156 and Q by S at position 158;-   N by N at position 156 and Q by T at position 158;-   L by N at position 157 and E by S at position 159;-   L by N at position 157 and E by T at position 159;-   Q by N at position 158 and S by S at position 160;-   Q by N at position 158 and S by T at position 160;-   E by N at position 159 and L by S at position 161;-   E by N at position 159 and L by T at position 161;-   S by N at position 160 and R by S at position 162;-   S by N at position 160 and R by T at position 162;-   L by N at position 161 and S by S at position 163;-   L by N at position 161 and S by T at position 163;-   R by N at position 162 and K by S at position 164;-   R by N at position 162 and K by T at position 164;-   S by N at position 163 and E by S at position 165; and-   S by N at position 163 and E by T at position 165,

where residue 1 corresponds to residue 1 of the mature IFNα-2b orIFNα-2a protein set forth in SEQ ID NO:1 or SEQ ID NO:182, respectively.In particular embodiments, the IFNα-2b or IFNα-2a mutant protein hasincreased resistance to proteolysis compared to unmodified IFNα-2b orIFNα-2a, and is selected from among proteins comprising one or more setsof duo-amino acid replacements in SEQ ID NO:1, corresponding to:

-   Q by N at position 5 and H by S at position 7;-   P by N at position 39 and E by S at position 41;-   P by N at position 39 and E by T at position 41;-   Q by N at position 40 and E by S at position 42;-   Q by N at position 40 and E by T at position 42;-   E by N at position 41 and F by S at position 43;-   E by N at position 41 and F by T at position 43;-   F by N at position 43 and N by S at position 45;-   G by N at position 44 and Q by T at position 46;-   N by N at position 45 and F by S at position 47;-   N by N at position 45 and F by T at position 47;-   Q by N at position 46 and Q by S at position 48;-   F by N at position 47 and K by S at position 49;-   F by N at position 47 and K by T at position 49;-   I by N at position 100 and G by S at position 102;-   I by N at position 100 and G by T at position 102;-   V by N at position 105 and E by S at position 107;-   V by N at position 105 and E by T at position 107;-   T by N at position 106 and T by S at position 108;-   T by N at position 106 and T by T at position 108;-   E by N at position 107 and P by S at position 109;-   E by N at position 107 and P by T at position 109;-   L by N at position 157 and E by S at position 159;-   L by N at position 157 and E by T at position 159;-   E by N at position 159 and L by S at position 161; and-   E by N at position 159 and L by T at position 161.    F. Protein Redesign

Provided herein are methods for designing and generating new versions ofnative or modified cytokines, such as IFNα-2b and IFNα-2a. Using thesemethods, the redesigned cytokine maintains either sufficient, typicallyequal or improved levels of a selected phenotype, such as a biologicalactivity, of the original protein, while at the same time its amino acidsequence is changed by replacement of up to: at least 1%, at least 2%,at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, atleast 8%, at least 9%, at least 10%, at least 12%, at least 14%, atleast 16%, at least 18%, at least 20%, at least 30%, at least 40% up to50% or more of its native amino acids by the appropriate pseudo-wildtype amino acids. Pseudo-wild type amino acids are those amino acidssuch that when they replace an original, such as native, amino acid at agiven position on the protein sequence, the resulting protein displayssubstantially the same levels of biological activity (or sufficientactivity for its therapeutic or other use) compared to the original,such as native, protein. In other embodiments, pseudo-wild type aminoacids are those amino acids such that when they replace an original,such as native, amino acid at a given position on the protein sequence,the resulting protein displays the same phenotype, such as levels ofbiological activity, compared to an original, typically a native,protein. Pseudo-wild type amino acids and the appropriate replacingpositions can be detected and identified by any analytical or predictivemeans; such as for example, by performing an alanine-scanning. Any otheramino acid, particularly another amino acid that has a neutral effect onstructure, such as Gly or Ser, also can be used for the scan. All thosereplacements of original, such as native, amino acids by Ala that do notlead to the generation of a HIT (a protein that has lost the desiredbiological activity), have either led to the generation of a LEAD (aprotein with increased biological activity); or the replacement by Alawill be a neutral replacement, i.e., the resulting protein will displaycomparable levels of biological activity compared to the original, suchas native, protein. The methods provided herein for protein redesign ofcytokines, such as IFNα-2b and IFNα-2a, are intended to design andgenerate “artificial” (versus naturally existing) proteins, such thatthey consist of amino acid sequences not existing in nature, but thatdisplay biological activities characteristic of the original, such asnative, protein. These redesigned proteins are contemplated herein to beuseful for avoiding potential side effects that might otherwise exist inother forms of cytokines in treatment of disease. Other uses ofredesigned proteins provided herein are to establish cross-talk betweenpathways triggered by different proteins; to facilitate structuralbiology by generating mutants that can be crystallized while maintainingactivity; and to destroy an activity of a protein without changing asecond activity or multiple additional activities.

In one embodiment, a method for obtaining redesigned proteins includesi) identifying some or all possible target sites on the protein sequencethat are susceptible to amino acid replacement without losing proteinactivity (protein activity in a largest sense of the term: enzymatic,binding, hormone, etc.) (These sites are the pseudo-wild type, ψ-wtsites); ii) identifying appropriate replacing amino acids (ψ-wt aminoacids), specific for each ψ-wt site, such that if used to replace thenative amino acids at that specific ψ-wt site, they can be expected togenerate a protein with comparable biological activity compared to theoriginal, such as native, protein, thus keeping the biological activityof the protein substantially unchanged; iii) systematically introducingthe specific ψ-wt amino acids at every specific ψ-wt position so as togenerate a collection containing the corresponding mutant molecules.Mutants are generated, produced and phenotypically characterizedone-by-one, in addressable arrays, such that each mutant moleculecontains initially amino acid replacements at only one ψ-wt site. Insubsequent rounds mutant molecules also can be generated such that theycontain one or more ψ-wt amino acids at one or more ψ-wt sites. Thosemutant proteins carrying several mutations at a number of ψ-wt sites,and that display comparable or improved biological activity are calledredesigned proteins or ψ-wt proteins. In particular embodiments, atleast 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%,at least 20%, at least 25%, or more of the amino acid residue positionson a particular cytokine, such as IFNα-2b and IFNα-2a are replaced withan appropriate pseudo-wild type amino acid.

The first step is an amino acid scan over the full length of theprotein. At this step, each and every one of the amino acids in theprotein sequence is replaced by a selected reference amino acid, such asalanine. This permits the identification of “redesign-HIT” positions,i.e., positions that are sensitive to amino acid replacement. All of theother positions that are not redesign-HIT positions (i.e., those atwhich the replacement of the original, such as native, amino acid by thereplacing amino acid, for example Ala, does not lead to a drop inprotein fitness or biological activity) are referred to herein as“pseudo-wild type” positions. When the replacing amino acid, for exampleAla, replaces the original, such as native, amino acid at a non-HITposition, then the replacement is neutral, in terms of protein activity,and the replacing amino acid is said to be a pseudo-wild type amino acidat that position. Pseudo-wild type positions appear to be less sensitivethan redesign-HIT positions since they tolerate the amino acidreplacement without affecting the protein activity that is being eithermaintained or improved. Amino acid replacement at the pseudo-wild typepositions, result in a non-change in the protein fitness (e.g., possesssubstantially the same biological activity), while at the same time to adivergence in the resulting protein sequence compared to the original,such as native, sequence.

To first identify those amino acid positions on the IFNα-2b and IFNα-2aprotein that are involved or not involved in IFNα-2b and IFNα-2a proteinactivity, such as binding activity of IFNα-2b and IFNα-2a to itsreceptor, an Ala-scan was performed on the IFNα-2b sequence as set forthin Example 4. For this purpose, each amino acid in the IFNα-2b proteinsequence was individually changed to Alanine. Any other amino acid,particularly another amino acid that has a neutral effect on structure,such as Gly or Ser, also can be used. Each resulting mutant IFNα-2bprotein was then expressed and the activity of the interferon moleculewas then assayed. These particular amino acid positions, referred toherein as HITs would in principle not be suitable targets for amino acidreplacement to increase protein stability, because of their involvementin the recognition of IFN-receptor or in the downstream pathwaysinvolved in IFN activity. For the Ala-scanning, the biological activitymeasured for the IFNα-2b molecules was: i) their capacity to inhibitvirus replication when added to permissive cells previously infectedwith the appropriate virus and, ii) their capacity to stimulate cellproliferation when added to the appropriate cells. The relative activityof each individual mutant compared to the native protein is indicated inFIG. 10A through C. HITs are those mutants that produce a decrease inthe activity of the protein (in the example: all the mutants withactivities below about 30% of the native activity.

In addition, the alanine-scan was used to identify the amino acidresidues on IFNα-2b that when replaced with alanine correspond to“pseudo-wild type” activity, i.e., those that can be replaced by alaninewithout leading to a decrease in biological activity. Knowledge of theseamino acids is useful for the re-design of the IFNα-2b and IFNα-2aproteins. The results are set forth in Table 5, and include pseudo-wildtype amino acid positions of IFNα-2b corresponding to SEQ ID NO:1, aminoacid residues: 9, 10, 17, 20, 24, 25, 35, 37, 41, 52, 54, 56, 57, 58,60, 63, 64, 65, 76, 89, and 90.

Accordingly, provided herein are IFNα-2b and IFNα-2a mutant proteinscomprising one or more pseudo-wild type mutations at amino acidpositions of IFNα-2b or IFNα-2a corresponding to SEQ ID NO:1 or SEQ IDNO:182, respectively, amino acid residues: 9, 10, 17, 20, 24, 25, 35,37, 41, 52, 54, 56, 57, 58, 60, 63, 64, 65, 76, 89, and 90. Themutations can be either one or more of insertions, deletions and/orreplacements of the native amino acid residue(s). In one embodiment, thepseudo-wild type replacements are mutations with alanine at eachposition. In another embodiment, the pseudo-wild type replacements areone or more mutations in SEQ ID NO:1 corresponding to:

L by A at position 9, L by A at position 17,

Q by A at position 20, I by A at position 24,

S by A at position 25, D by A at position 35,

G by A at position 37, E by A at position 41,

T by A at position 52, P by A at position 54,

L by A at position 56, H by A at position 57,

E by A at position 58, I by A at position 60,

I by A at position 63, F by A at position 64,

N by A at position 65, W by A at position 76,

Y by A at position 89, and Q by A at position 90.

In addition, the IFNα-2b alanine scan revealed the followingredesign-HITs having decreased antiviral activity at amino acidpositions of IFNα-2b corresponding to SEQ ID NO:1, amino acid residues:2, 7, 8, 11, 13, 15, 16, 23, 26, 28, 29, 30, 31, 32, 33, 53, 69, 91, 93,98, and 101. Accordingly, in particular embodiments where it is desiredto decrease the anti-viral activity of IFNα-2b or IFNα-2a, either one ormore of insertions, deletions and/or replacements of the native aminoacid residue(s) can be carried out at one or more of amino acidpositions of IFNα-2b or IFNα-2a corresponding to SEQ ID NO:1, amino acidresidues: 2, 7, 8, 11, 13, 15, 16, 23, 26, 28, 29, 30, 31, 32, 33, 53,69, 91, 93, 98, and 101.

Each of the redesign mutations set forth above can be combined with oneor more of the IFNα-2b or IFNα-2a candidate LEAD mutations or one ormore of the IFNα-2b or IFNα-2a LEAD mutants provided herein.

G. 3D-Scanning and Its Use for Modifying Cytokines

Also provided herein is a method of structural homology analysis forcomparing proteins regardless of their underlying amino acid sequences.For a subset of proteins families, such as the family of humancytokines, this information is rationally exploited to produce modifiedproteins. This method of structural homology analysis can be applied toproteins that are evolved by any method, including the 2D scanningmethod described herein. When used with the 2D method in which aparticular phenotype, activity or characteristic of a protein ismodified by 2D analysis, the method is referred to as 3D-scanning.

The use of “structural homology” analysis in combination with thedirected evolution methods provided herein provides a powerful techniquefor identifying and producing various new protein mutants, such ascytokines, having desired biological activities, such as increasedresistance to proteolysis. For example, the analysis of the “structuralhomology” between an optimized mutant version of a given protein and“structurally homologous” proteins allows identification of thecorresponding structurally related or structurally similar amino acidpositions (also referred to herein as “structurally homologous loci”) onother proteins. This permits identification of mutant versions of thelatter that have a desired optimized feature(s) (biological activity,phenotype) in a simple, rapid and predictive manner (regardless of aminoacid sequence and sequence homology). Once a mutant version of a proteinis developed, then, by applying the rules of structural homology, thecorresponding structurally related amino acid positions (and replacingamino acids) on other “structurally homologous” proteins readily areidentified, thus allowing a rapid and predictive discovery of theappropriate mutant versions for the new proteins.

3-dimensionally structurally equivalent or similar amino acid positionsthat are located on two or more different protein sequences that share acertain degree of structural homology, have comparable functional tasks(activities and phenotypes). These two amino acids that occupysubstantially equivalent 3-dimensional structural space within theirrespective proteins than can be said to be “structurally similar” or“structurally related” with each other, even if their precise positionson the amino acid sequences, when these sequences are aligned, do notmatch with each other. The two amino acids also are said to occupy“structurally homologous loci.” “Structural homology” does not take intoaccount the underlying amino acid sequence and solely compares3-dimensional structures of proteins. Thus, two proteins can be said tohave some degree of structural homology whenever they shareconformational regions or domains showing comparable structures orshapes with 3-dimensional overlapping in space. Two proteins can be saidto have a higher degree of structural homology whenever they share ahigher amount of conformational regions or domains showing comparablestructures or shapes with 3-dimensional overlapping in space. Aminoacids positions on one or more proteins that are “structurallyhomologous” can be relatively far way from each other in the proteinsequences, when these sequences are aligned following the rules ofprimary sequence homology. Thus, when two or more protein backbones aredetermined to be structurally homologous, the amino acid residues thatare coincident upon three-dimensional structural superposition arereferred to as “structurally similar” or “structurally related” aminoacid residues in structurally homologous proteins (also referred to as“structurally homologous loci”). Structurally similar amino acidresidues are located in substantially equivalent spatial positions instructurally homologous proteins.

For example, for proteins of average size (approximately 180 residues),two structures with a similar fold will usually display rms deviationsnot exceeding 3 to 4 angstroms. For example, structurally similar orstructurally related amino acid residues can have backbone positionsless than 3.5, 3.0, 2.5, 2.0, 1.7 or 1.5 angstrom from each other uponprotein superposition. RMS deviation calculations and proteinsuperposition can be carried out using any of a number of methods knownin the art. For example, protein superposition and RMS deviationcalculations can be carried out using all peptide backbone atoms (e.g.,N, C, C(C═O), O and CA (when present)). As another example, proteinsuperposition can be carried out using just one or any combination ofpeptide backbone atoms, such as, for example, N, C, C(C═O), O and CA(when present). In addition, one skilled in the art will recognize thatprotein superposition and RMS deviation calculations generally can beperformed on only a subset of the entire protein structure. For example,if the protein superposition is carried out using one protein that hasmany more amino acid residues than another protein, proteinsuperposition can be carried out on the subset (e.g., a domain) of thelarger protein that adopts a structure similar to the smaller protein.Similarly, only portions of other proteins can be suitable forsuperimposition. For example, if the position of the C-terminal residuesfrom two structurally homologous proteins differ significantly, theC-terminal residues can be omitted from the structural superposition orRMS deviation calculations.

Accordingly, provided herein are methods of rational evolution ofproteins based on the identification of potential target sites formutagenesis (is-HITs) through comparison of patterns of protein backbonefolding between structurally related proteins, irrespective of theunderlying sequences of the compared proteins. Once the structurallyrelated amino acid positions are identified on the new protein, thensuitable amino acid replacement criteria, such as PAM analysis, can beemployed to identify candidate LEADs for construction and screening asdescribed herein.

For example, analysis of “structural homology” between and among anumber of related cytokines was used to identify on various members ofthe cytokine family, other than interferon alpha, those amino acidpositions and residues that are structurally similar or structurallyrelated to those found in the IFNα-2b mutants provided herein that havebeen optimized for improved stability. The resulting modified cytokinesare provided. This method can be applied to any desired phenotype usingany protein, such as a cytokine, as the starting material to which anevolution procedure, such as the rational directed evolution procedureof U.S. application Ser. No. 10/022,249 or the 2-dimensional scanningmethod provided herein, is applied. The structurally correspondingresidues are then altered on members of the family to produce additionalcytokines with similar phenotypic alterations.

1. Homology

Typically, homology between proteins is compared at the level of theiramino acid sequences, based on the percent or level of coincidence ofindividual amino acids, amino acid per amino acid, when sequences arealigned starting from a reference, generally the residue encoded by thestart codon. For example, two proteins are said to be “homologous” or tobear some degree of homology whenever their respective amino acidsequences show a certain degree of matching upon alignment comparison.Comparative molecular biology is primarily based on this approach. Fromthe degree of homology or coincidence between amino acid sequences,conclusions can be made on the evolutionary distance between or amongtwo or more protein sequences and biological systems.

The concept of “convergent evolution” is applied to describe thephenomena by which phylogenetically unrelated organisms or biologicalsystems have evolved to share features related to their anatomy,physiology and structure as a response to common forces, constraints,and evolutionary demands from the surrounding environment and livingorganisms. Alternatively, “divergent evolution,” is applied to describethe phenomena by which strongly phylogenetically related organisms orbiological systems have evolved to diverge from identity or similarityas a response to divergent forces, constraints, and evolutionary demandsfrom the surrounding environment and living organisms.

In the typical traditional analysis of homologous proteins there are twoconceptual biases corresponding to: i) “convergent evolution,” and ii)“divergent evolution.” Whenever the aligned amino acid sequences of twoproteins do not match well with each other, these proteins areconsidered “not related” or “less related” with each other and havedifferent phylogenetic origins. There is no (or low) homology betweenthese proteins and their respective genes are not homologous (or showlittle homology). If these two “non-homologous” proteins under studyshare some common functional features (e.g., interaction with otherspecific molecules, activity), they are determined to have arisen by“convergent evolution,” i.e., by evolution of their non-homologous aminoacid sequences, in such a way that they end up generating functionally“related” structures.

On the other hand, whenever the aligned amino acid sequences of twoproteins do match with each other to a certain degree, these proteinsare considered to be “related” and to share a common phylogeneticorigin. A given degree of homology is assigned between these twoproteins and their respective genes likewise share a correspondingdegree of homology. During the evolution of their initial highlyhomologous amino acid sequence, enough changes can be accumulated insuch a way that they end up generating “less-related” sequences and lessrelated function. The divergence from perfect matching between these two“homologous” proteins under study is said come from “divergentevolution.”

2. 3D-Scanning (Structural Homology) Methods

Structural homology refers to homology between the topology andthree-dimensional structure of two proteins. Structural homology is notnecessarily related to “convergent evolution” or to “divergentevolution,” nor is it related to the underlying amino acid sequence.Rather, structural homology is likely driven (through natural evolution)by the need of a protein to fit specific conformational demands imposedby its environment. Particular structurally homologous “spots” or “loci”would not be allowed to structurally diverge from the originalstructure, even when its own underlying sequence does diverge. Thisstructural homology is exploited herein to identify loci for mutation.

Within the amino acid sequence of a protein resides the appropriatebiochemical and structural signals to achieve a specific spatial foldingin either an independent or a chaperon-assisted manner. Indeed, thisspecific spatial folding ultimately determines protein traits andactivity. Proteins interact with other proteins and molecules in generalthrough their specific topologies and spatial conformations. Inprinciple, these interactions are not based solely on the precise aminoacid sequence underlying the involved topology or conformation. Ifprotein traits, activity (behavior and phenotypes) and interactions relyon protein topology and conformation, then evolutionary forces andconstraints acting on proteins can be expected to act on topology andconformation. Proteins sharing similar functions will share comparablecharacteristics in their topology and conformation, despite theunderlying amino acid sequences that create those topologies andconformations.

3. Application of the 3D-Scanning Methods to Cytokines

The method based on structural homology, including the 3D-scanningmethod provided herein can be applied to any related proteins. Forexemplary purposes herein it is applied to cytokines. In exemplaryembodiments, methods for altering phenotypes of members of families ofcytokines by altering one member such as by employing the 2-dimensionalrational scanning method are provided. As provided herein, other membersof these cytokine families then can be similarly modified by identifyingand changing structurally homologous residues to similarly alter thephenotypes of such proteins.

In an exemplary embodiment herein, IFNα-2b mutants with increasedresistance to proteolysis are generated by the 2-dimensional rationalscanning method; IFNβ mutants also were generated. The correspondingresidues on members of cytokine families that possess structuralhomology to IFNα-2b were identified and the identified residues on theother cytokines were similarly modified to produce cytokines withincreased resistance to proteolysis. Hence also provided herein arecytokine mutants that display increased resistance to proteolysis and/orglomerular filtration containing one or more amino acid replacements.

Provided herein are mutant (modified) cytokines that display alteredfeatures and properties, such as a resistance to proteolysis. Methodsfor producing such modified cytokines also are provided.

Also provided herein is a method of structural homology analysis forcomparing proteins regardless their underlying amino acid sequences. Fora subset of proteins families, such as the family of human cytokines,this information is rationally exploited herein. Human cytokines allshare a common helix bundle fold, which is used to structurally definethe 4-helical cytokine superfamily in the structural classification ofthe protein database SCOP© (Structural Classification of Proteins; see,e.g., Murzin et al., J. Mol. Biol., 247:536-540, 1995 and“scop.mrc-lmb.cam.ac.uk/scop/”). This superfamily includes threedifferent families: 1) the interferons/interleukin-10 protein family(SEQ ID NOS: 1 and 182-200); 2) the long-chain cytokine family (SEQ IDNOS: 210-217); and 3) the short-chain cytokine family (SEQ ID NOS:201-209).

For example, a distinct feature of cytokines from theinterferons/interleukin-10 family is an additional (fifth) helix. Thisfamily includes interleukin-10 (IL-10; SEQ ID NO:200, interferon beta(IFNβ; SEQ ID NO: 196), interferon alpha-2a (IFNα-2a; SEQ ID NO: 182),interferon alpha-2b (IFNα-2b; SEQ ID NO:1), and interferon gamma (IFN-γ,SEQ ID NO: 199). The long-chain cytokine protein family includes, amongothers, granulocyte colony stimulating factor (G-CSF; SEQ ID NO: 210),leukemia inhibitory factor (LIF; SEQ ID NO: 213), growth hormone (hGH;SEQ ID NO: 216), ciliary neurotrophic factor (CNTF; SEQ ID NO: 212),leptin (SEQ ID NO: 211), oncostatin M (SEQ ID NO: 214), interleukin-6(IL-6; SEQ ID NO: 217) and interleukin-12 (IL-12; SEQ ID NO: 215). Theshort-chain cytokine protein family includes, among others,erythropoietin (EPO; SEQ ID NO: 201), granulocyte-macrophage colonystimulating factor (GM-CSF; SEQ ID NO: 202), interleukin-2 (IL-2; SEQ IDNO: 204), interleukin-3 (IL-3; SEQ ID NO: 205), interleukin-4 (IL-4; SEQID NO: 207), interleukin-5 (IL-5; SEQ ID NO: 208), interleukin-13(IL-13; SEQ ID NO: 209), Flt3 ligand (SEQ ID NO: 203) and stem cellfactor (SCF; SEQ ID NO: 206).

Although the degree of similarity among the underlying amino acidsequences of these cytokines does not appear high, their corresponding3-dimensional structures present a high level of similarity (see, e.g.,FIGS. 8B through D). Effectively, the best structural similarity isobtained between two 3-dimensional protein structures of the same familyin the 4-helical cytokine superfamily.

The methods provided herein for producing mutant cytokines areexemplified with reference to production of cytokines that display asubstantially equivalent increase in resistance to proteolysis relativeto the optimized IFNα-2b mutants. It is understood that this method canbe applied to other families of proteins and for other phenotypes.

In one embodiment, proteins of the 4-helical cytokine superfamily areprovided herein that are structurally homologous IFNα-2b LEAD mutantsset forth herein. For example, by virtue of the knowledge of the3-dimensional structural amino acid positions within the LEAD IFNα-2bmutants provided herein that confer higher resistance to a challengewith either proteases or blood lysate or serum, while maintaining orimproving the requisite biological activity, the correspondingstructurally related (e.g., structurally similar) amino acid residues ona variety of other cytokines are identified (FIG. 9).

Numerous methods are well known in the art for identifying structurallyrelated amino acid positions with 3-dimensionally structurallyhomologous proteins. Exemplary methods include, but are not limited to:CATH (Class, Architecture, Topology and Homologous superfamily) which isa hierarchical classification of protein domain structures based on fourdifferent levels (Orengo et al., Structure, 5(8):1093-1108 (1997)); CE(Combinatorial Extension of the optimal path), which is a method thatcalculates pairwise structure alignments (Shindyalov et al., ProteinEngineering, 11(9):739-747 (1998)); FSSP (Fold classification based onStructure-Structure alignment of Proteins), which is a database based onthe complete comparison of all 3-dimensional protein structures thatcurrently reside in the Protein Data Bank (PDB) (Holm et al., Science,273:595-602 (1996)); SCOP® (Structural Classification of Proteins),which provides a descriptive database based on the structural andevolutionary relationships between all proteins whose structure is known(Murzin et al., J. Mol. Biol., 247:536-540 (1995)); and VAST (VectorAlignment Search Tool), which compares newly determined 3-dimensionalprotein structure coordinates to those found in the MMDB/PDB database(Gibrat et al., Current Opinion in Structural Biology, 6:377-385(1995)).

In an exemplary embodiment, the step-by-step process including the useof a program referred to as TOP (see FIG. 8A and Lu, G., J. Appl.Cryst., 33:176-189 (2000)); publicly available, for example, atbioinfo1.mbfys.lu.se/TOP is used for protein structure comparison. Thisprogram runs two steps for each protein structure comparison. In thefirst step topology of secondary structure in the two structures iscompared. The program uses two points to represent each secondarystructure element (alpha helices or beta strands) then systematicallysearches all the possible super-positions of these elements in3-dimensional space (defined as the root mean square deviation—rmsd, theangle between the two lines formed by the two points and the line-linedistance). The program searches to determine whether additionalsecondary structure elements can fit by the same superpositionoperation. If secondary structures that can fit each other exceed agiven number, the program identifies the two structures as similar. Theprogram gives as an output a comparison score called “StructuralDiversity” that considers the distance between matched α-carbon atomsand the number of matched residues. The lower the “Structural Diversity”score, the more the two structures are similar. In various embodimentsherein, the Structural Diversity scores range from 0 up to about 67.

In the exemplified embodiment, all the cytokines were first structurallyaligned against the IFNα-2b structure. For the proteins within the samefamily as IFNα-2b (e.g., the interferons/interleukin-10 cytokinefamily), this alignment was directly used to identify the structurallyrelated is-HIT target amino acid positions and/or regions correspondingto the structurally homologous positions and/or regions on IFNα-2b whereLEAD mutants were found (FIG. 8B). For the other cytokines, the proteinof the family (either long- or short-chain cytokines) with the best3-dimensional structural alignment with IFNα-2b was selected using thelowest “Structural Diversity” score as the representative for thatfamily. From the short-chain cytokine protein family, erythropoietin(EPO; see FIG. 8C) was identified as the best structural homologue ofIFNα-2b (rmsd=1.9 angstroms; number of aligned residues=62; StructuralDiversity=13.8). From the long-chain cytokine protein family,granulocyte-colony stimulating factor (G-CSF; see FIG. 8D) wasidentified as the best structural homologue of IFNα-2b (rmsd=1.7angstroms; number of aligned residues=77; Structural Diversity=7.8).

Next, the amino acid positions and/or regions corresponding to the LEADmutant regions on IFNα-2b were identified on these two proteins. Thesetwo best structural homologues of IFNα-2b (e.g., EPO and G-CSF; seeFIGS. 12L and 12E, respectively) were structurally aligned to each ofthe other cytokines within their respective cytokine protein families.As a result, protein regions likely to be targets for serum proteaseresistance were identified on all cytokines (see FIGS. 12A through T).Amino acids in these target regions were then checked for their exposureto the solvent and their susceptibility to be protease substrate.Exposed and substrate residues are then subjected to PAM250 analysis asset forth above, so that a group of non-substrate and functionallyconservative amino acid residues are selected as replacements. Theresults of the above structural homology analysis for each of thecytokines provided herein are set forth in FIGS. 12A through T.

Accordingly, provided herein are modified cytokines that exhibit greaterresistance to proteolysis compared to the unmodified cytokine protein,comprising one or more amino acid replacements at one or more targetpositions on the cytokine corresponding to a structurally-relatedmodified amino acid position within the 3-dimensional structure of anIFNα-2b modified protein provided herein. The resistance to proteolysiscan be measured by mixing it with a protease in vitro, incubation withblood or incubation with serum. Also provided herein are cytokinestructural homologues of an IFNα-2b modified protein provided herein,comprising one or more amino acid replacements in the cytokinestructural homologue at positions corresponding to the3-dimensional-structurally-similar modified positions within the3-dimensional structure of the modified IFNα-2b. In one embodiment, thecytokine homologue has increased resistance to proteolysis compared toits unmodified and/or wild type cytokine counterpart. Resistance toproteolysis can be measured by mixture with a protease in vitro,incubation with blood, or incubation with serum.

a. Structurally Homologous Interferon Mutants

Also provided herein are modified cytokines or cytokine structuralhomologues of IFNα-2b that are IFNα cytokines. These IFNα cytokinesinclude, but are not limited to, IFNα-2a, IFNα-c, IFNα-2c, IFNα-d,IFNα-5, IFNα-6, IFNα-4, IFNα-4b, IFNα-I, IFNα-J, IFNα-H, IFNα-F, IFNα-8and IFNα-consensus cytokine (see, SEQ ID No. 232). Accordingly, amongthe modified IFNα cytokines provided herein are those with one or moreamino acid replacements at one or more target positions in eitherIFNα-2a, IFNα-c, IFNα-2c, IFNα-d, IFNα-5, IFNα-6, IFNα-4, IFNα-4b,IFNα-I, IFNα-J, IFNα-H, IFNα-F, IFNα-8, or IFNα-consensus cytokinecorresponding to a structurally-related modified amino acid positionwithin the 3-dimensional structure of the IFNα-2b modified proteinsprovided herein. The replacements lead to greater resistance toproteases, as assessed by incubation with a protease or a with a bloodlysate or by incubation with serum, compared to the unmodified IFNα-2a.

In particular embodiments, the modified IFNα cytokines are selected fromamong:

the modified IFNα-2a that is human and is selected from among proteinscomprising one or more single amino acid replacements in SEQ ID NO: 182,corresponding to amino acid positions: 41, 58, 78, 107, 117, 125, 133and 159;

the modified IFNα-c that is human and is selected from among proteinscomprising one or more single amino acid replacements in SEQ ID NO: 183,corresponding to amino acid positions: 41, 59, 79, 108, 118, 126, 134and 160;

the modified IFNα-2c cytokine that is human and is selected from amongcytokines comprising one or more single amino acid replacements in SEQID NO: 185, corresponding to amino acid positions: 41, 58, 78, 107, 117,125, 133 and 159;

the IFNα-d modified protein that is human and is selected from amongproteins comprising one or more single amino acid replacements in SEQ IDNO: 186, corresponding to amino acid positions: 41, 59, 79, 108, 118,126, 134 and 160;

the IFNα-5 modified protein that is human and is selected from amongproteins comprising one or more single amino acid replacements in SEQ IDNO: 187, corresponding to amino acid positions: 41, 59, 79, 108, 118,126, 134 and 160;

the IFNα-6 modified protein that is human and is selected from amongproteins comprising one or more single amino acid replacements in SEQ IDNO: 188, corresponding to amino acid positions: 41, 59, 79, 108, 118,126, 134 and 160;

the IFNα-4 modified protein that is human and is selected from amongproteins comprising one or more single amino acid replacements in SEQ IDNO: 189, corresponding to amino acid positions: 41, 59, 79, 108, 118,126, 134 and 160;

the IFNα-4b modified protein that is human and is selected from amongproteins comprising one or more single amino acid replacements in SEQ IDNO: 190, corresponding to amino acid positions: 41, 59, 79, 108, 118,126, 134 and 160;

the IFNα-I modified protein that is human and is selected from amongproteins comprising one or more single amino acid replacements in SEQ IDNO: 191, corresponding to amino acid positions: 41, 59, 79, 108, 118,126, 134 and 160;

the IFNα-J modified protein that is human and is selected from amongproteins comprising one or more single amino acid replacements in SEQ IDNO: 192, corresponding to amino acid positions: 41, 59, 79, 108, 118,126, 134 and 160;

the IFNα-H modified protein that is human and is selected from amongproteins comprising one or more single amino acid replacements in SEQ IDNO: 193, corresponding to amino acid positions: 41, 59, 79, 108, 118,126, 134 and 160;

the IFNα-F modified protein that is human and is selected from amongproteins comprising one or more single amino acid replacements in SEQ IDNO: 194, corresponding to amino acid positions: 41, 59, 79, 108, 118,126, 134 and 160;

the IFNα-8 modified protein that is human and is selected from amongproteins comprising one or more single amino acid replacements in SEQ IDNO: 195, corresponding to amino acid positions: 41, 59, 79, 108, 118,126, 134 and 160; and

the IFNα-consensus modified protein that is human and is selected fromamong proteins that contain one or more single amino acid replacementsin SEQ ID NO: 232, corresponding to amino acid positions: 41, 58, 78,107, 117, 125, 133 and 159.

b. Structurally Homologous Cytokine Mutants

As set forth above, provided herein are modified cytokines that containone or more amino acid replacements at one or more target positions ineither interleukin-10 (IL-10), interferon beta (IFNβ, IFNβ-1, IFNβ-2a,interferon gamma (IFN-γ), granulocyte colony stimulating factor (G-CSF),and human erythropoietin (EPO); corresponding to a structurally-relatedmodified amino acid position within the 3-dimensional structure of theIFNα-2b modified proteins provided herein. The replacements lead togreater resistance to proteases, as assessed by incubation with aprotease or a with a blood lysate or by incubation with serum, comparedto the unmodified cytokine.

Also provided herein are modified cytokines that contain one or moreamino acid replacements at one or more target positions in eithergranulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2(IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5(IL-5), interleukin-13 (IL-13), Flt3 ligand and stem cell factor (SCF);corresponding to a structurally-related modified amino acid positionwithin the 3-dimensional structure of the human EPO modified proteinsprovided herein. The replacements lead to greater resistance toproteases, as assessed by incubation with a protease or a with a bloodlysate or by incubation with serum, compared to the unmodified cytokine.

Also provided herein are modified cytokines that contain one or moreamino acid replacements at one or more target positions in eitherinterleukin-10 (IL-10), interferon beta (IFNβ), interferon gamma(IFN-γ), human granulocyte colony stimulating factor (G-CSF), leukemiainhibitory factor (LIF), human growth hormone (hGH), ciliaryneurotrophic factor (CNTF), leptin, oncostatin M, interleukin-6 (IL-6)and interleukin-12 (IL-12); corresponding to a structurally-relatedmodified amino acid position within the 3-dimensional structure of thehuman G-CSF modified proteins provided herein. The replacements lead togreater resistance to proteases, as assessed by incubation with aprotease or a with a blood lysate or by incubation with serum, comparedto the unmodified cytokine.

In particular embodiments, the modified cytokines are selected from thefollowing:

A modified IFNβ cytokine, comprising mutations at one or more amino acidresidues of IFNβ corresponding to SEQ ID NO: 196: 39, 42, 45, 47, 52,67, 71, 73, 81, 107, 108, 109, 110, 111, 113, 116, 120, 123, 124, 128,130, 134, 136, 137, 163 and 165. The mutations include insertions,deletions and replacements of the native amino acid residue(s). Inparticular embodiments, the replacements are selected from among aminoacid substitutions in SEQ ID NO: 196 set forth in FIG. 12A correspondingto SEQ ID NOS: 233-289, where the first amino acid indicated issubstituted by the second at the position indicated for all of thesubstitutions set forth in FIG. 12A through T.

A modified IFN-gamma cytokine, comprising mutations at one or more aminoacid residues of IFN-gamma corresponding to SEQ ID NO:199: 33, 37, 40,41, 42, 58, 61, 64, 65 and 66. The mutations include insertions,deletions and replacements of the native amino acid residue(s). Inparticular embodiments, the replacements are selected from among aminoacid substitutions in SEQ ID NO:199 set forth in FIG. 12B correspondingto SEQ ID NOS: 290-311.

A modified IL-10 cytokine, comprising mutations at one or more aminoacid residues of IL-10 corresponding to SEQ ID NO:200: 49, 50, 52, 53,54, 55, 56, 57, 59, 60, 67, 68, 71, 72, 74, 75, 78, 81, 84, 85, 86, and88. The mutations include insertions, deletions and replacements of thenative amino acid residue(s). In exemplary embodiments, replacements areselected from among amino acid substitutions in SEQ ID NO:200 set forthin FIG. 12C corresponding to SEQ ID NOS: 312-361.

A modified erythropoietin cytokine, comprising mutations at one or moreamino acid residues of erythropoietin corresponding to SEQ ID NO:201:43, 45, 48, 49, 52, 53, 55, 72, 75, 76, 123, 129, 130, 131, 162, and165. The mutations include insertions, deletions and replacements of thenative amino acid residue(s). In exemplary embodiments, the replacementsare selected from among amino acid substitutions in SEQ ID NO: 201 setforth in FIG. 12L corresponding to SEQ ID NOS: 940-977.

A modified GM-CSF cytokine, comprising mutations at one or more aminoacid residues of GM-CSF corresponding to SEQ ID NO: 202: 38, 41, 45, 46,48, 49, 51, 60, 63, 67, 92, 93, 119, 120, 123, and 124. The mutationsinclude insertions, deletions and replacements of the native amino acidresidue(s). In exemplary embodiments, the replacements are selected fromamong amino acid substitutions in SEQ ID NO: 202 set forth in FIG. 12Ncorresponding to SEQ ID NOS: 362-400.

A modified Flt3 ligand cytokine, comprising mutations at one or moreamino acid residues of Flt3 ligand corresponding to SEQ ID NO: 203: 3,40, 42, 43, 55, 58, 59, 61, 89, 90, 91, 95, and 96. The mutationsinclude insertions, deletions and replacements of the native amino acidresidue(s). In exemplary embodiments, the replacements are selected fromamong amino acid substitutions in SEQ ID NO: 203 set forth in FIG. 12Mcorresponding to SEQ ID NOS: 401-428.

A modified IL-2 cytokine, comprising mutations at one or more amino acidresidues of IL-2 corresponding to SEQ ID NO: 204 at positions 43, 45,48, 49, 52, 53, 60, 61, 65, 67, 68, 72, 100, 103, 104, 106, 107, 109,110, and 132. The mutations include insertions, deletions andreplacements of the native amino acid residue(s). In exemplaryembodiments, the replacements are selected from among amino acidsubstitutions in SEQ ID NO: 204 set forth in FIG. 12P and SEQ ID NOS:429-476.

A modified IL-3 cytokine, comprising mutations at one or more amino acidresidues of IL-3 corresponding to SEQ ID NO: 205: 37, 43, 46, 59, 63,66, 96, 100, 101, and 103. The mutations include insertions, deletionsand replacements of the native amino acid residue(s). In exemplaryembodiments, the replacements are selected from among amino acidsubstitutions in SEQ ID NO:205 set forth in FIG. 12Q corresponding toSEQ ID NOS: 477-498.

A modified SCF cytokine, comprising mutations at one or more amino acidresidues of SCF corresponding to SEQ ID NO: 206: 27, 31, 34, 37, 54, 58,61, 62, 63, 96, 98, 99, 100, 102, 103, 106, 107, 108, 109, 134, and 137.The mutations include insertions, deletions and replacements of thenative amino acid residue(s). In exemplary embodiments, the replacementsare selected from among amino acid substitutions in SEQ ID NO: 206 setforth in FIG. 12T corresponding to SEQ ID NOS: 499-542.

A modified IL-4 cytokine, comprising mutations at one or more amino acidresidues of IL-4 corresponding to SEQ ID NO: 207: 26, 37, 53, 60, 61,64, 66, 100, 102, 103, and 126. The mutations include insertions,deletions and replacements of the native amino acid residue(s). Inexemplary embodiments, the replacements are selected from among aminoacid substitutions in SEQ ID NO: 207 set forth in FIG. 12R correspondingto SEQ ID NOS: 543-567.

A modified IL-5 cytokine, comprising mutations at one or more amino acidresidues of IL-5 corresponding to SEQ ID NO: 208: 32, 34, 39, 46, 47,56, 84, 85, 88, 89, 90, 102, 110, and 111. The mutations includeinsertions, deletions and replacements of the native amino acidresidue(s). In exemplary embodiments, the replacements are selected fromamong amino acid substitutions in SEQ ID NO: 208 set forth in FIG. 12Scorresponding to SEQ ID NOS: 568-602.

A modified IL-13 cytokine, comprising mutations at one or more aminoacid residues of IL-13 corresponding to SEQ ID NO: 209: 32, 34, 38, 48,79, 82, 85, 86, 88, 107, 108, 110, and 111. The mutations includeinsertions, deletions and replacements of the native amino acidresidue(s). In exemplary embodiments, the replacements are selected fromamong amino acid substitutions in SEQ ID NO: 209 set forth in FIG. 12Ocorresponding to SEQ ID NOS: 603-630.

A modified G-CSF cytokine, comprising mutations at one or more aminoacid residues of G-CSF corresponding to SEQ ID NO: 210: 61, 63, 68, 72,86, 96, 100, 101, 131, 133, 135, 147, 169, 172, and 177. The mutationsinclude insertions, deletions and replacements of the native amino acidresidue(s). In exemplary embodiments, the replacements are selected fromamong amino acid substitutions in SEQ ID NO: 210 set forth in FIG. 12Ecorresponding to SEQ ID NOS: 631-662.

A modified leptin cytokine, comprising mutations at one or more aminoacid residues of leptin corresponding to SEQ ID NO: 211: 43, 49, 99,100, 104, 105, 107, 108, 141 and 142. The mutations include insertions,deletions and replacements of the native amino acid residue(s). Inexemplary embodiments, the replacements are selected from among aminoacid substitutions in SEQ ID NO: 211 set forth in FIG. 12I correspondingto SEQ ID NOS: 663-683.

A modified CNTF cytokine, comprising mutations at one or more amino acidresidues of CNTF corresponding to SEQ ID NO: 212: 62, 64, 66, 67, 86,89, 92, 100, 102, 104, 131, 132, 133, 135, 136, 138, 140, 143, 148, and151. The mutations include insertions, deletions and replacements of thenative amino acid residue(s). In exemplary embodiments, the replacementsare selected from among amino acid substitutions in SEQ ID NO: 212 setforth in FIG. 12D corresponding to SEQ ID NOS: 684-728.

A modified LIF cytokine, comprising mutations at one or more amino acidresidues of LIF corresponding to SEQ ID NO: 213: 69, 70, 85, 99, 102,104, 106, 109, 137, 143, 146, 148, 149, 153, 154, and 156. The mutationsinclude insertions, deletions and replacements of the native amino acidresidue(s). In exemplary embodiments, the replacements are selected fromamong amino acid substitutions in SEQ ID NO: 213 set forth in FIG. 12Jcorresponding to SEQ ID NOS: 729-760.

A modified oncostatin M cytokine, comprising mutations at one or moreamino acid residues of oncostatin M corresponding to SEQ ID NO: 214: 59,60, 63, 65, 84, 87, 89, 91, 94, 97, 99, 100, 103, and 106. The mutationsinclude insertions, deletions and replacements of the native amino acidresidue(s). In exemplary embodiments, the replacements are selected fromamong amino acid substitutions in SEQ ID NO: 214 set forth in FIG. 12Kcorresponding to SEQ ID NOS: 761-793.

A modified IL-12 cytokine, comprising mutations at one or more aminoacid residues of IL-12 corresponding to SEQ ID NO: 215: 56, 61, 66, 67,68, 70, 72, 75, 78, 79, 82, 89, 92, 93, 107, 110, 111, 115, 117, 124,125, 127, 128, 129, and 189. The mutations include insertions, deletionsand replacements of the native amino acid residue(s). In exemplaryembodiments, the replacements are selected from among amino acidsubstitutions in SEQ ID NO: 215 set forth in FIG. 12G corresponding toSEQ ID NOS: 794-849.

A modified hGH cytokine, comprising mutations at one or more amino acidresidues of hGH corresponding to SEQ ID NO: 216: 56, 59, 64, 65, 66, 88,92, 94, 101, 129, 130, 133, 134, 140, 143, 145, 146, 147, 183, and 186.The mutations include insertions, deletions and replacements of thenative amino acid residue(s). In exemplary embodiments, the replacementsare selected from among amino acid substitutions in SEQ ID NO: 216 setforth in FIG. 12F corresponding to SEQ ID NOS: 850-895.

A modified IL-6 cytokine, comprising mutations at one or more amino acidresidues of IL-6 corresponding to SEQ ID NO: 217: 64, 65, 66, 68, 69,75, 77, 92, 98, 103, 105, 108, 133, 138, 139, 140, 149, 156, 178, and181. The mutations include insertions, deletions and replacements of thenative amino acid residue(s). In exemplary embodiments, the replacementsare selected from among amino acid substitutions in SEQ ID NO: 217 setforth in FIG. 12H corresponding to SEQ ID NOS: 896-939.

In certain embodiments, the modified cytokines provided herein possessincreased stability compared to the unmodified cytokine. Stability canbe assessed by any in vitro or in vivo method, such as by measuringresidual inhibition of viral replication or to stimulation of cellproliferation in appropriate cells, after incubation with eithermixtures of proteases, individual proteases, blood lysate or serum.

In other embodiments, the modified cytokines provided herein possessdecreased stability compared to the unmodified cytokine. Stability canbe assessed by any in vitro or in vivo method, such as by measuringresidual inhibition of viral replication or to stimulation of cellproliferation in appropriate cells, after incubation with eithermixtures of proteases, individual proteases, blood lysate or serum.

In other embodiments, the modified cytokines provided herein possessincreased activity compared to the unmodified cytokine. Stability can beassessed by any in vitro or in vivo method, such as by measuringresidual inhibition of viral replication or to stimulation of cellproliferation in appropriate cells, after incubation with eithermixtures of proteases, individual proteases, blood lysate or serum.

H. Rational Evolution of IFNβ for Increased Resistance to Proteolysisand/or Higher Conformational Stability

Treatment with interferon β (IFNβ) is a well established therapy.Typically it is used for treatment of multiple sclerosis (MS). Patientsreceiving interferon β are subject to frequent repeat applications ofthe drug. The instability of IFNβ in the blood stream and under thestorage conditions is well known. Hence it would be useful to increasingstability (half-life) of IFNβ in serum and also in vitro would improveit as a drug.

The 2D-scanning method and the 3D-scanning method (using structuralhomology) provided herein (see, copending U.S. application Ser. No.10/658,355, filed Sep. 8, 2003, based on U.S. provisional applicationSer. Nos. 60/457,063 and 60/410,258) were each applied to interferon β.Provided herein are mutant variants of the IFNβ protein that displayimproved stability as assessed by resistance to proteases (therebypossessing increased protein half-life) and at least comparablebiological activity as assessed by antiviral or antiproliferationactivity compared to the unmodified and wild type native IFNβ protein(SEQ ID NO: 196). The IFNβ mutant proteins provided herein confer ahigher half-life and at least comparable biological activity withrespect to the native sequence. Thus, the optimized IFNβ protein mutantsprovided herein that possess increased resistance to proteolysis resultin a decrease in the frequency of injections needed to maintain asufficient drug level in serum, thus leading to, for example: i) highercomfort and acceptance by patients, ii) lower doses necessary to achievecomparable biological effects, and iii) as a consequence of (ii), likelyattenuation of any secondary effects.

In exemplary embodiments, the half-life of the IFNβ mutants providedherein is increased by an amount selected from at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 100%, at least 150%, at least200%, at least 250%, at least 300%, at least 350%, at least 400%, atleast 450%, at least 500% or more, when compared to the half-life ofnative human IFNβ in either human blood, human serum or an in vitromixture containing one or more proteases. In other embodiments, thehalf-life of the IFNβ mutants provided herein is increased by an amountselected from at least 6 times, 7 times, 8 times, 9 times, 10 times, 20times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times,700 times, 800 times, 900 times, 1000 times, or more, when compared tothe half-life of native human IFNβ in either human blood, human serum oran in vitro mixture containing one or more proteases.

Two approaches were used herein to increase the stability of IFNβ byamino acid replacement: i) Resistance to proteases: amino acidreplacement that leads to higher resistance to proteases by directdestruction of the protease target residue or sequence, while eithermaintaining or improving the requisite biological activity (e.g.,antiviral and anti-proliferation activity), and/or ii) conformationalstability: amino acid replacement that leads to an increase inconformational stability (i.e. half-life at room temperature or at 37°C.), while either improving or maintaining the requisite biologicalactivity (e.g., antiviral and anti-proliferation activity).

Two methodologies were used to address the improvements described above:(a) 2D-scanning methods were used to identify amino acid changes thatlead to improvement in protease resistance and to improvement inconformational stability, and (b) 3D-scanning, which employs structuralhomology methods also were used to identify amino acid changes that leadto improvement in protease resistance. The 2D-scanning and 3D-scanningmethods each were used to identify the amino acid changes on IFNβ thatlead to an increase in stability when challenged either with proteases,human blood lysate or human serum. Increasing protein stability toproteases, human blood lysate or human serum is contemplated herein toprovide a longer in vivo half-life for the particular protein molecules,and thus a reduction in the frequency of necessary injections intopatients. The biological activities that have been measured for the IFNβmolecules are i) their capacity to inhibit virus replication when addedto permissive cells previously infected with the appropriate virus, andii) their capacity to stimulate cell proliferation when added to theappropriate cells. Prior to the measurement of biological activity, IFNβmolecules were challenged with proteases, human blood lysate or humanserum during different incubation times. The biological activitymeasured, corresponds then to the residual biological activity followingexposure to the proteolytic mixtures.

As set forth above, provided herein are methods for the generating IFNβmolecules (or any target protein, particularly cytokines) that, whilemaintaining a requisite biological activity without substantial change(sufficient for therapeutic application(s)), have been rendered lesssusceptible to digestion by blood proteases and therefore display alonger half-life in blood circulation. In this particular example, themethod used included the following specific steps as exemplified in theExamples:

For the improvement of resistance to proteases, by 2D-scanning, themethod included:

1) Identifying some or all possible target sites on the protein sequencethat are susceptible to digestion by one or more specific proteases(these sites are the is-HITs); and

2) Identifying appropriate replacing amino acids, specific for eachis-HIT, such that if used to replace one or more of the original aminoacids at that specific is-HIT, they can be expected to increase theis-HIT's resistance to digestion by protease while at the same time,keeping the biological activity of the protein unchanged (thesereplacing amino acids are the candidate LEADs).

For the improvement of resistance to proteases, by 3D-scanning(structural homology):

1) Identifying some or all possible target sites (is-HITS) on theprotein sequence that display an acceptable degree of structuralhomology around the amino acid positions mutated in the LEAD moleculespreviously obtained for IFNα using 2D-scanning, and that are susceptibleto digestion by one or more specific proteases; and

2) Identifying appropriate replacing amino acids, specific for eachis-HIT, such that if used to replace one or more of the original aminoacids at that specific is-HIT, they can be expected to increase theis-HIT's resistance to digestion by protease while at the same time,keeping the biological activity of the protein unchanged (thesereplacing amino acids are the candidate LEADs).

For the improvement of conformational stability, by 2D-scanning, asprovided herein:

-   -   1) Identifying some or all possible target sites on the protein        sequence that are susceptible to being directly involved in the        intramolecular flexibility and conformational change (these        sites are the is-HITs); and    -   2) Identifying appropriate replacing amino acids, specific for        each is-HIT, such that if used to replace one or more of the        original amino acids at that specific is-HIT, they can be        expected to increase the thermal stability of the molecule while        at the same time, keeping the biological activity of the protein        unchanged (these replacing amino acids are the candidate LEADs).        See FIGS. 6(O)-6(S) and FIG. 8(A).

Using the 2D-scanning and 3D-scanning methods and the 3-dimensionalstructure of IFNβ, the following amino acid target positions wereidentified as is-HITs on IFNβ, which numbering is that of the matureprotein (SEQ ID NO:196):

By 3D-scanning (see, SEQ ID Nos.: 234-289, 989-1015): D by Q at position39, D by H at position 39, D by G at position 39, E by Q at position 42,E by H at position 42, K by Q at position 45, K by T at position 45, Kby S at position 45, K by H at position 45, L by V at position 47, L byI at position 47, L by T at position 47, L by Q at position 47, L by Hat position 47, L by A at position 47, K by Q at position 52, K by T atposition 52, K by S at position 52, K by H at position 52, F by I atposition 67, F by V at position 67, R by H at position 71, R by Q atposition 71, D by H at position 73, D by G at position 73, D by Q atposition 73, E by Q at position 81, E by H at position 81, E by Q atposition 107, E by H at position 107, K by Q at position 108, K by T atposition 108, K by S at position 108, K by H at position 108, E by Q atposition 109, E by H at position 109, D by Q at position 110, D by H atposition 110, D by G at position 110, F by I at position 111, F by V atposition 111, R by H at position 113, R by Q at position 113, L by V atposition 116, L by I at position 116, L by T at position 116, L by Q atposition 116, L by H at position 116, L by A at position 116, L by V atposition 120, L by I at position 120, L by T at position 120, L by Q atposition 120, L by H at position 120, L by A at position 120, K by Q atposition 123, K by T at position 123, K by S at position 123, K by H atposition 123, R by H at position 124, R by Q at position 124, R by H atposition 128, R by Q at position 128, L by V at position 130, L by I atposition 130, L by T at position 130, L by Q at position 130, L by H atposition 130, L by A at position 130, K by Q at position 134, K by T atposition 134, K by S at position 134, K by H at position 134, K by Q atposition 136, K by T at position 136, K by S at position 136, K by H atposition 136, E by Q at position 137, E by H at position 137, Y by H atposition 163, Y by I at position 163, R by H at position 165, R by Q atposition 165.

By 2D-scanning (see SEQ ID Nos.: 1016-1302): M by V at position 1, M byI at position 1, M by T at position 1, M by Q at position 1, M by A atposition 1, L by V at position 5, L by I at position 5, L by T atposition 5, L by Q at position 5, L by H at position 5, L by A atposition 5, F by I at position 8, F by V at position 8, L by V atposition 9, L by I at position 9, L by T at position 9, L by Q atposition 9, L by H at position 9, L by A at position 9, R by H atposition 11, R by Q at position 11, F by I at position 15, F by V atposition 15, K by Q at position 19, K by T at position 19, K by S atposition 19, K by H at position 19, W by S at position 22, W by H atposition 22, N by H at position 25, N by S at position 25, N by Q atposition 25, R by H position 27, R by Q position 27, L by V at position28, L by I at position 28, L by T at position 28, L by Q at position 28,L by H at position 28, L by A at position 28, E by Q at position 29, Eby H at position 29, Y by H at position 30, Y by I at position 30, L byV at position 32, L by I at position 32, L by T at position 32, L by Qat position 32, L by H at position 32, L by A at position 32, K by Q atposition 33, K by T at position 33, K by S at position 33, K by H atposition 33, R by H at position 35, R by Q at position 35, M by V atposition 36, M by I at position 36, M by T at position 36, M by Q atposition 36, M by A at position 36, D by Q at position 39, D by H atposition 39, D by G at position 39, E by Q at position 42, E by H atposition 42, K by Q at position 45, K by T at position 45, K by S atposition 45, K by H at position 45, L by V at position 47, L by I atposition 47, L by T at position 47, L by, Q at position 47, L by H atposition 47, L by A at position 47, K by Q at position 52, K by T atposition 52, K by S at position 52, K by H at position 52, F by I atposition 67, F by V at position 67, R by H at position 71, R by Q atposition 71, D by Q at position 73, D by H at position 73, D by G atposition 73, E by Q at position 81, E by H at position 81, E by Q atposition 85, E by H at position 85, Y by H at position 92, Y by I atposition 92, K by Q at position 99, K by T at position 99, K by S atposition 99, K by H at position 99, E by Q at position 103, E by H atposition 103, E by Q at position 104, E by H at position 104, K by Q atposition 105, K by T at position 105, K by S at position 105, K by H atposition 105, E by Q at position 107, E by H at position 107, K by Q atposition 108, K by T at position 108, K by S at position 108, K by H atposition 108, E by Q at position 109, E by H at position 109, D by Q atposition 110, D by H at position 110, D by G at position 110, F by I atposition 111, F by V at position 111, R by H at position 113, R by Q atposition 113, L by V at position 116, L by I at position 116, L by T atposition 116, L by Q at position 116, L by H at position 116, L by A atposition 116, L by V at position 120, L by I at position 120, L by T atposition 120, L by Q at position 120, L by H at position 120, L by A atposition 120, K by Q at position 123, K by T at position 123, K by S atposition 123, K by H at position 123, R by H at position 124, R by Q atposition 124, R by H at position 128, R by Q at position 128, L by V atposition 130, L by I at position 130, L by T at position 130, L by Q atposition 130, L by H at position 130, L by A at position 130, K by Q atposition 134, K by T at position 134, K by S at position 134, K by H atposition 134, K by Q at position 136, K by T at position 136, K by S atposition 136, K by H at position 136, E by Q at position 137, E by H atposition 137, Y by H at position 138, Y by I at position 138, R by H atposition 152, R by Q at position 152, Y by H at position 155, Y by I atposition 155, R by H at position 159, R by Q at position 159, Y by H atposition 163, Y by I at position 163, R by H at position 165, R by Q atposition 165, M by D at position 1, M by E at position 1, M by K atposition 1, M by N at position 1, M by R at position 1, M by S atposition 1, L by D at position 5, L by E at position 5, L by K atposition 5, L by N at position 5, L by R at position 5, L by S atposition 5, L by D at position 6, L by E at position 6, L by K atposition 6, L by N at position 6, L by R at position 6, L by S atposition 6, L by Q at position 6, L by T at position 6, F by E atposition 8, F by K at position 8, F by R at position 8, F by D atposition 8, L by D at position 9, L by E at position 9, L by K atposition 9, L by N at position 9, L by R at position 9, L by S atposition 9, Q by D at position 10, Q by E at position 10, Q by K atposition 10, Q by N at position 10, Q by R at position 10, Q by S atposition 10, Q by T at position 10, S by D at position 12, S by E atposition 12, S by K at position 12, S by R at position 12, S by D atposition 13, S by E at position 13, S by K at position 13, S by R atposition 13, S by N at position 13, S by Q at position 13, S by T atposition 13, N by D at position 14, N by E at position 14, N by K atposition 14, N by Q at position 14, N by R at position 14, N by S atposition 14, N by T at position 14, F by D at position 15, F by E atposition 15, F by K at position 15, F by R at position 15, Q by D atposition 16, Q by E at position 16, Q by K at position 16, Q by N atposition 16, Q by R at position 16, Q by S at position 16, Q by T atposition 16, C by D at position 17, C by E at position 17, C by K atposition 17, C by N at position 17, C by Q at position 17, C by R atposition 17, C by S at position 17, C by T at position 17, L by N atposition 20, L by Q at position 20, L by R at position 20, L by S atposition 20, L by T at position 20, L by D at position 20, L by E atposition 20, L by K at position 20, W by D at position 22, W by E atposition 22, W by K at position 22, W by R at position 22, Q by D atposition 23, Q by E at position 23, Q by K at position 23, Q by R atposition 23, L by D at position 24, L by E at position 24, L by K atposition 24, L by R at position 24, W by D at position 79, W by E atposition 79, W by K at position 79, W by R at position 79, N by D atposition 80, N by E at position 80, N by K at position 80, N by R atposition 80, T by D at position 82, T by E at position 82, T by K atposition 82, T by R at position 82, I by D at position 83, I by E atposition 83, I by K at position 83, I by R at position 83, I by N atposition 83, I by Q at position 83, I by S at position 83, I by T atposition 83, N by D at position 86, N by E at position 86, N by K atposition 86, N by R at position 86, N by Q at position 86, N by S atposition 86, N by T at position 86, L by D at position 87, L by E atposition 87, L by K at position 87, L by R at position 87, L by N atposition 87, L by Q at position 87, L by S at position 87, L by T atposition 87, A by D at position 89, A by E at position 89, A by K atposition 89, A by R at position 89, N by D at position 90, N by E atposition 90, N by K at position 90, N by Q at position 90, N by R atposition 90, N by S at position 90, N by T at position 90, V by D atposition 91, V by E at position 91, V by K at position 91, V by N atposition 91, V by Q at position 91, V by R at position 91, V by S atposition 91, V by T at position 91, Q by D at position 94, Q by E atposition 94, Q by Q at position 94, Q by N at position 94, Q by R atposition 94, Q by S at position 94, Q by T at position 94, I by D atposition 95, I by E at position 95, I by K at position 95, I by N atposition 95, I by Q at position 95, I by R at position 95, I by S atposition 95, I by T at position 95, H by D at position 97, H by E atposition 97, H by K at position 97, H by N at position 97, H by Q atposition 97, H by R at position 97, H by S at position 97, H by T atposition 97, L by D at position 98, L by E at position 98, L by K atposition 98, L by N at position 98, L by Q at position 98, L by R atposition 98, L by S at position 98, L by T at position 98, V by D atposition 101, V by E at position 101, V by K at position 101, V by N atposition 101, V by Q at position 101, V by R at position 101, V by S atposition 101, V by T at position 101, M by C at position 1, L by C atposition 6, Q by C at position 10, S by C at position 13, Q by C atposition 16, L by C at position 17, V by C at position 101, L by C atposition 98, H by C at position 97, Q by C at position 94, V by C atposition 91, N by C at position 90.

SEQ ID NO. Mutant SEQ ID No 1016 (M1V) SEQ ID No 1017 (M1I) SEQ ID No1018 (M1T) SEQ ID No 1019 (M1A) SEQ ID No 1020 (L5V) SEQ ID No 1021(L5I) SEQ ID No 1022 (L5T) SEQ ID No 1023 (L5Q) SEQ ID No 1024 (L5H) SEQID No 1025 (L5A) SEQ ID No 1026 (F8I) SEQ ID No 1027 (F8V) SEQ ID No1028 (L9V) SEQ ID No 1029 (L9I) SEQ ID No 1030 (L9T) SEQ ID No 1031(L9Q) SEQ ID No 1032 (L9H) SEQ ID No 1033 (L9A) SEQ ID No 1034 (R11H)SEQ ID No 1035 (R11Q) SEQ ID No 1036 (F15I) SEQ ID No 1037 (F15V) SEQ IDNo 1038 (K19Q) SEQ ID No 1039 (K19T) SEQ ID No 1040 (K19S) SEQ ID No1041 (K19H) SEQ ID No 1042 (W22S) SEQ ID No 1043 (W22H) SEQ ID No 1044(N25H) SEQ ID No 1045 (N25S) SEQ ID No 1046 (N25Q) SEQ ID No 1047 (R27H)SEQ ID No 1048 (R27Q) SEQ ID No 1049 (L28V) SEQ ID No 1050 (L28I) SEQ IDNo 1051 (L28T) SEQ ID No 1052 (L28Q) SEQ ID No 1053 (L28H) SEQ ID No1054 (L28A) SEQ ID No 1055 (E29Q) SEQ ID No 1056 (E29H) SEQ ID No 1057(Y30H) SEQ ID No 1058 (Y30I) SEQ ID No 1059 (L32V) SEQ ID No 1060 (L32I)SEQ ID No 1061 (L32T) SEQ ID No 1062 (L32Q) SEQ ID No 1063 (L32H) SEQ IDNo 1064 (L32A) SEQ ID No 1065 (M1Q) SEQ ID No 1066 (K33Q) SEQ ID No 1067(K33T) SEQ ID No 1068 (K33S) SEQ ID No 1069 (K33H) SEQ ID No 1070 (R35H)SEQ ID No 1071 (R35Q) SEQ ID No 1072 (M36V) SEQ ID No 1073 (M36I) SEQ IDNo 1074 (M36T) SEQ ID No 1075 (M36Q) SEQ ID No 1076 (M36A) SEQ ID No1077 (E85Q) SEQ ID No 1078 (E85H) SEQ ID No 1079 (Y92H) SEQ ID No 1080(Y92I) SEQ ID No 1081 (K99Q) SEQ ID No 1082 (K99T) SEQ ID No 1083 (K99S)SEQ ID No 1084 (K99H) SEQ ID No 1085 (E103Q) SEQ ID No 1086 (E103H) SEQID No 1087 (E104Q) SEQ ID No 1088 (E104H) SEQ ID No 1089 (K105Q) SEQ IDNo 1090 (K105T) SEQ ID No 1091 (K105S) SEQ ID No 1092 (K105H) SEQ ID No1093 (Y138H) SEQ ID No 1094 (Y138I) SEQ ID No 1095 (R152H) SEQ ID No1096 (R152Q) SEQ ID No 1097 (Y155H) SEQ ID No 1098 (Y155I) SEQ ID No1099 (R159H) SEQ ID No 1100 (R159Q) SEQ ID No 1101 (M1D) SEQ ID No 1102(M1E) SEQ ID No 1103 (M1K) SEQ ID No 1104 (M1N) SEQ ID No 1105 (M1R) SEQID No 1106 (M1S) SEQ ID No 1107 (L5D) SEQ ID No 1108 (L5E) SEQ ID No1109 (L5K) SEQ ID No 1110 (L5R) SEQ ID No 1111 (L5N) SEQ ID No 1112(L5S) SEQ ID No 1113 (L6D) SEQ ID No 1114 (L6E) SEQ ID No 1115 (L6K) SEQID No 1116 (L6N) SEQ ID No 1117 (L6Q) SEQ ID No 1118 (L6R) SEQ ID No1119 (L6S) SEQ ID No 1120 (L6T) SEQ ID No 1121 (F8D) SEQ ID No 1122(F8E) SEQ ID No 1123 (F8K) SEQ ID No 1124 (F8R) SEQ ID No 1125 (L9D) SEQID No 1126 (L9E) SEQ ID No 1127 (L9K) SEQ ID No 1128 (L9N) SEQ ID No1129 (L9R) SEQ ID No 1130 (L9S) SEQ ID No 1131 (Q10D) SEQ ID No 1132(Q10E) SEQ ID No 1133 (Q10K) SEQ ID No 1134 (Q10N) SEQ ID No 1135 (Q10R)SEQ ID No 1136 (Q10S) SEQ ID No 1137 (Q10T) SEQ ID No 1138 (S12D) SEQ IDNo 1139 (S12E) SEQ ID No 1140 (S12K) SEQ ID No 1141 (S12R) SEQ ID No1142 (S13D) SEQ ID No 1143 (S13E) SEQ ID No 1144 (S13K) SEQ ID No 1145(S13N) SEQ ID No 1146 (S13Q) SEQ ID No 1147 (S13R) SEQ ID No 1148 (S13T)SEQ ID No 1149 (N14D) SEQ ID No 1150 (N14E) SEQ ID No 1151 (N14K) SEQ IDNo 1152 (N14Q) SEQ ID No 1153 (N14R) SEQ ID No 1154 (N14S) SEQ ID No1155 (N14T) SEQ ID No 1156 (F15D) SEQ ID No 1157 (F15E) SEQ ID No 1158(F15K) SEQ ID No 1159 (F15R) SEQ ID No 1160 (Q16D) SEQ ID No 1161 (Q16E)SEQ ID No 1162 (Q16K) SEQ ID No 1163 (Q16N) SEQ ID No 1164 (Q16R) SEQ IDNo 1165 (Q16S) SEQ ID No 1166 (Q16T) SEQ ID No 1167 (C17D) SEQ ID No1168 (C17E) SEQ ID No 1169 (C17K) SEQ ID No 1170 (C17N) SEQ ID No 1171(C17Q) SEQ ID No 1172 (C17R) SEQ ID No 1173 (C17S) SEQ ID No 1174 (C17T)SEQ ID No 1175 (L20N) SEQ ID No 1176 (L20Q) SEQ ID No 1177 (L20R) SEQ IDNo 1178 (L20S) SEQ ID No 1179 (L20T) SEQ ID No 1180 (L20D) SEQ ID No1181 (L20E) SEQ ID No 1182 (L20K) SEQ ID No 1183 (W22D) SEQ ID No 1184(W22E) SEQ ID No 1185 (W22K) SEQ ID No 1186 (W22R) SEQ ID No 1187 (Q23D)SEQ ID No 1188 (Q23E) SEQ ID No 1189 (Q23K) SEQ ID No 1190 (Q23R) SEQ IDNo 1191 (L24D) SEQ ID No 1192 (L24E) SEQ ID No 1193 (L24K) SEQ ID No1194 (L24R) SEQ ID No 1195 (G78D) SEQ ID No 1196 (G78E) SEQ ID No 1197(G78K) SEQ ID No 1198 (G78R) SEQ ID No 1199 (W79D) SEQ ID No 1200 (W79E)SEQ ID No 1201 (W79K) SEQ ID No 1202 (W79R) SEQ ID No 1203 (N80D) SEQ IDNo 1204 (N80E) SEQ ID No 1205 (N80K) SEQ ID No 1206 (N80R) SEQ ID No1207 (T82D) SEQ ID No 1208 (T82E) SEQ ID No 1209 (T82K) SEQ ID No 1210(T82R) SEQ ID No 1211 (I83D) SEQ ID No 1212 (I83E) SEQ ID No 1213 (I83K)SEQ ID No 1214 (I83R) SEQ ID No 1215 (I83N) SEQ ID No 1216 (I83Q) SEQ IDNo 1217 (I83S) SEQ ID No 1218 (I83T) SEQ ID No 1219 (N86D) SEQ ID No1220 (N86E) SEQ ID No 1221 (N86K) SEQ ID No 1222 (N86R) SEQ ID No 1223(N86Q) SEQ ID No 1224 (N86S) SEQ ID No 1225 (N86T) SEQ ID No 1226 (L87D)SEQ ID No 1227 (L87E) SEQ ID No 1228 (L87K) SEQ ID No 1229 (L87R) SEQ IDNo 1230 (L87N) SEQ ID No 1231 (L87Q) SEQ ID No 1232 (L87S) SEQ ID No1233 (L87T) SEQ ID No 1234 (A89D) SEQ ID No 1235 (A89E) SEQ ID No 1236(A89K) SEQ ID No 1237 (A89R) SEQ ID No 1238 (N90D) SEQ ID No 1239 (N90E)SEQ ID No 1240 (N90K) SEQ ID No 1241 (N90Q) SEQ ID No 1242 (N90R) SEQ IDNo 1243 (N90S) SEQ ID No 1244 (N90T) SEQ ID No 1245 (V91D) SEQ ID No1246 (V91E) SEQ ID No 1247 (V91K) SEQ ID No 1248 (V91N) SEQ ID No 1249(V91Q) SEQ ID No 1250 (V91R) SEQ ID No 1251 (V91S) SEQ ID No 1252 (V91T)SEQ ID No 1253 (Q94D) SEQ ID No 1254 (Q94E) SEQ ID No 1255 (Q94K) SEQ IDNo 1256 (Q94N) SEQ ID No 1257 (Q94R) SEQ ID No 1258 (Q94S) SEQ ID No1259 (Q94T) SEQ ID No 1260 (I95D) SEQ ID No 1261 (I95E) SEQ ID No 1262(I95K) SEQ ID No 1263 (I95N) SEQ ID No 1264 (I95Q) SEQ ID No 1265 (I95R)SEQ ID No 1266 (I95S) SEQ ID No 1267 (I95T) SEQ ID No 1268 (H97D) SEQ IDNo 1269 (H97E) SEQ ID No 1270 (H97K) SEQ ID No 1271 (H97N) SEQ ID No1272 (H97Q) SEQ ID No 1273 (H97R) SEQ ID No 1274 (H97S) SEQ ID No 1275(H97T) SEQ ID No 1276 (L98D) SEQ ID No 1277 (L98E) SEQ ID No 1278 (L98K)SEQ ID No 1279 (L98N) SEQ ID No 1280 (L98Q) SEQ ID No 1281 (L98R) SEQ IDNo 1282 (L98S) SEQ ID No 1283 (L98T) SEQ ID No 1284 (V101D) SEQ ID No1285 (V101E) SEQ ID No 1286 (V101K) SEQ ID No 1287 (V101N) SEQ ID No1288 (V101Q) SEQ ID No 1289 (V101R) SEQ ID No 1290 (V101S) SEQ ID No1291 (V101T) SEQ ID No 1292 (M1C) SEQ ID No 1293 (V101C) SEQ ID No 1294(L6C) SEQ ID No 1295 (L98C) SEQ ID No 1296 (Q10C) SEQ ID No 1297 (H97C)SEQ ID No 1298 (S13C) SEQ ID No 1299 (Q94C) SEQ ID No 1300 (Q16C) SEQ IDNo 1301 (N90C) SEQ ID No 1302 (V91C)I. Super-Leads and Additive Directional Mutagenesis (ADM)

Also provided herein are super-LEAD mutant proteins comprising acombination of single amino acid mutations present in two or more of therespective LEAD mutant proteins. Thus, the super-LEAD mutant proteinshave two of more of the single amino acid mutations derived from two ormore of the respective LEAD mutant proteins. As described herein, LEADmutant proteins provided herein are defined as mutants whose performanceor fitness has been optimized with respect to the native protein. LEADstypically contain one single mutation relative to its respective nativeprotein. This mutation represents an appropriate amino acid replacementthat takes place at one is-HIT position. Further super-LEAD mutantproteins are created such that they carry on the same protein molecule,more than one LEAD mutation, each at a different is-HIT position. Oncethe LEAD mutant proteins have been identified using the 2D-scanningmethods provided herein, super-LEADs can be generated by combining twoor more individual LEAD mutant mutations using methods well-known in theart, such as recombination, mutagenesis and DNA shuffling, and bymethods, such as additive directional mutagenesis and Multi-OverlappedPrimer Extensions, provided herein.

1. Additive Directional Mutagenesis

Also provided herein are methods for assembling on a single mutantprotein multiple mutations present on the individual LEAD molecules, soas to generate super-LEAD mutant proteins. This method is referred toherein as “Additive Directional Mutagenesis” (ADM). ADM is a repetitivemulti-step process where at each step after the creation of the firstLEAD mutant protein a new LEAD mutation is added onto the previous LEADmutant protein to create successive super-LEAD mutant proteins. ADM isnot based on genetic recombination mechanisms, nor on shufflingmethodologies; instead it is a simple one-mutation-at-a-time process,repeated as many times as necessary until the total number of desiredmutations is introduced on the same molecule. To avoid the exponentiallyincreasing number of all possible combinations that can be generated byputting together on the same molecule a given number of singlemutations, a method is provided herein that, although it does not coverall the combinatorial possible space, still captures a big part of thecombinatorial potential. The word “combinatorial” is used here in itsmathematical meaning (i.e., subsets of a group of elements, containingsome of the elements in any possible order) and not in the molecularbiological or directed evolution meaning (i.e., generating pools, ormixtures, or collections of molecules by randomly mixing theirconstitutive elements).

A population of sets of nucleic acid molecules encoding a collection ofnew super-LEAD mutant molecules is generated, tested and phenotypicallycharacterized one-by-one in addressable arrays. super-LEAD mutantmolecules are such that each molecule contains a variable number andtype of LEAD mutations. Those molecules displaying further improvedfitness for the particular feature being evolved, are referred to assuper-LEADs. Super-LEADs may be generated by other methods known tothose of skill in the art and tested by the high throughput methodsherein. For purposes herein a super-LEAD typically has activity withrespect to the function or biological activity of interest that differsfrom the improved activity of a LEAD by a desired amount, such as atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% ormore from at least one of the LEAD mutants from which it is derived. Inyet other embodiments, the change in activity is at least about 2 times,3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times,20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times,700 times, 800 times, 900 times, 1000 times, or more greater than atleast one of the LEAD molecules from which it is derived. As with LEADs,the change in the activity for super-LEADs is dependent upon theactivity that is being “evolved.” The desired alteration, which can beeither an increase or a reduction in activity, will depend upon thefunction or property of interest.

In one embodiment provided herein, the ADM method employs a number ofrepetitive steps, such that at each step a new mutation is added on agiven molecule. Although numerous different ways are possible forcombining each LEAD mutation onto a super-LEAD protein, an exemplary waythe new mutations (e.g., mutation 1 (m1), mutation 2 (m2), mutation 3(m3), mutation 4 (m4), mutation 5 (m5), mutation n (mn)) can be addedcorresponds to the following diagram:

m1

m1+m2

m1+m2+m3

m1+m2+m3+m4

m1+m2+m3+m4+m5

m1+m2+m3+m4+m5+ . . . +mn

m1+m2+m4

m1+m2+m4+m5

m1+m2+m4+m5+ . . . +mn

m1+m2+m5

m1+m2+m5+ . . . +mn

m2

m2+m3

m2+m3+m4

m2+m3+m4+m5

m2+m3+m4+m5+ . . . +mn

m2+m4

m2+m4+m5

m2+m4+m5+ . . . +mn

m2+m5

m2+m5+ . . . +mn

. . . , etc. . . .

2. Multi-Overlapped Primer Extensions

In another embodiment, provided herein is a method for the rationalevolution of proteins using oligonucleotide-mediated mutagenesisreferred to as “multi overlapped primer extensions.” This method can beused for the rational combination of mutant LEADs to form super-LEADS.This method allows the simultaneous introduction of several mutationsthroughout a small protein or protein-region of known sequence.Overlapping oligonucleotides of typically around 70 bases in length(since longer oligonucleotides lead to increased error) are designedfrom the DNA sequence (gene) encoding the mutant LEAD proteins in such away that they overlap with each other on a region of typically around 20bases. These overlapping oligonucleotides (including or not pointmutations) act as both template and primers in a first step of PCR(using a proofreading polymerase, e.g., Pfu DNA polymerase, to avoidunexpected mutations) to create small amounts of full-length gene. Thefull-length gene resulting from the first PCR is then selectivelyamplified in a second step of PCR using flanking primers, each onetagged with a restriction site in order to facilitate subsequentcloning. One multi overlapped extension process yields a full-length(multi-mutated) nucleic acid molecule encoding a candidate super-LEADprotein having multiple mutations therein derived from LEAD mutantproteins.

Although typically about 70 bases are used to create the overlappingoligonucleotides, the length of additional overlapping oligonucleotidesfor use herein can range from about 30 bases up to about 100 bases, fromabout 40 bases up to about 90 bases, from about 50 bases up to about 80bases, from about 60 bases up to about 75 bases, and from about 65 basesup to about 75 bases. As set forth above, typically about 70 bases areused herein.

Likewise, although typically the overlapping region of the overlappingoligonucleotides is about 20 bases, the length of other overlappingregions for use herein can range from about 5 bases up to about 40bases, from about 10 bases up to about 35 bases, from about 15 bases upto about 35 bases, from about 15 bases up to about 25 bases, from about16 bases up to about 24 bases, from about 17 bases up to about 23 bases,from about 18 bases up to about 22 bases, and from about 19 bases up toabout 21 bases. As set forth above, typically about 20 bases are usedherein for the overlapping region.

J. Uses of the Mutant IFNα and IFNβ Genes and Cytokines in TherapeuticMethods

The optimized cytokines provided herein, such as the IFNα-2b and IFNβproteins and other modified cytokines, are intended for use in varioustherapeutic as well as diagnostic methods. These include all methods forwhich the unmodified proteins are used. By virtue of their improvedphenotypes and activities, the proteins provided herein should exhibitimprovement in the corresponding in vivo phenotype.

In particular, the optimized cytokines, such as the IFNα-2b and IFNβproteins, are intended for use in therapeutic methods in which cytokineshave been used for treatment. Such methods include, but are not limitedto, methods of treatment of infectious diseases, allergies, microbialdiseases, pregnancy related diseases, bacterial diseases, heartdiseases, viral diseases, histological diseases, genetic diseases, bloodrelated diseases, fungal diseases, adrenal diseases, cancers, liverdiseases, autoimmune diseases, growth disorders, diabetes,neurodegenerative diseases, including multiple sclerosis, Parkinson'sdisease and Alzheimer's disease.

1. Fusion Proteins

Fusion proteins containing a targeting agent and mutant IFNα, includingIFNα-2b and IFNα-2a, and IFNβ mutant proteins, or cytokine protein alsoare provided. Pharmaceutical compositions containing such fusionproteins formulated for administration by a suitable route are provided.Fusion proteins are formed by linking in any order the mutant proteinand an agent, such as an antibody or fragment thereof, growth factor,receptor, ligand and other such agent for directing the mutant proteinto a targeted cell or tissue. Linkage can be effected directly orindirectly via a linker. The fusion proteins can be producedrecombinantly or chemically by chemical linkage, such as viaheterobifunctional agents or thiol linkages or other such linkages. Thefusion proteins can contain additional components, such as E. colimaltose binding protein (MBP) that aid in uptake of the protein by cells(see, International PCT application No. WO 01/32711).

2. Nucleic Acid Molecules for Expression

Nucleic acid molecules encoding the mutant cytokines including themutant IFNβ proteins and IFN α proteins, such as the IFNα-2b and IFNα-2aproteins, provided herein, or the fusion protein operably linked to apromoter, such as an inducible promoter for expression in mammaliancells also are provided. Such promoters include, but are not limited to,CMV and SV40 promoters; adenovirus promoters, such as the E2 genepromoter, which is responsive to the HPV E7 oncoprotein; a PV promoter,such as the PBV p89 promoter that is responsive to the PV E2 protein;and other promoters that are activated by the HIV or PV or oncogenes.

The mutant cytokines including the mutant interferons (IFNα's andIFNβ's) proteins provided herein, also can be delivered to the cells ingene transfer vectors. The transfer vectors also can encode additionalother therapeutic agent(s) for treatment of the disease or disorder,such cancer or HIV infection, for which the cytokine is administered.

3. Formulation of Optimized Cytokines and Methods of Treatment

Pharmaceutical compositions containing an optimized cytokine producedherein, such as IFNα-2b, IFNα-2a and IFNβ, fusion proteins or encodingnucleic acid molecules can be formulated in any conventional manner bymixing a selected amount of an optimized cytokine with one or morephysiologically acceptable carriers or excipients. Selection of thecarrier or excipient depends upon the mode of administration (i.e.,systemic, local, topical or any other mode) and disorder treated. Thepharmaceutical compositions provided herein can be formulated for singledosage administration. The concentrations of the compounds in theformulations are effective for delivery of an amount, uponadministration, that is effective for the intended treatment. Typically,the compositions are formulated for single dosage administration. Toformulate a composition, the weight fraction of a compound or mixturethereof is dissolved, suspended, dispersed or otherwise mixed in aselected vehicle at an effective concentration such that the treatedcondition is relieved or ameliorated. Pharmaceutical carriers orvehicles suitable for administration of the compounds provided hereininclude any such carriers known to those skilled in the art to besuitable for the particular mode of administration.

In addition, the compounds may be formulated as the solepharmaceutically active ingredient in the composition or may be combinedwith other active ingredients. Liposomal suspensions, includingtissue-targeted liposomes, may also be suitable as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art. For example, liposome formulations may beprepared as described in U.S. Pat. No. 4,522,811.

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the compounds in known in vitro and in vivo systems, such as theassays provided herein. The active compounds can be administered by anyappropriate route, for example, orally, parenterally, intravenously,intradermally, subcutaneously, or topically, in liquid, semi-liquid orsolid form and are formulated in a manner suitable for each route ofadministration.

The optimized cytokine and physiologically acceptable salts and solvatescan be formulated for administration by inhalation (either through themouth or the nose) or for oral, buccal, parenteral or rectaladministration. For administration by inhalation, the optimized cytokinecan be delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluorethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator can be formulatedcontaining a powder mix of a therapeutic compound and a suitable powderbase such as lactose or starch.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions can take the form of tablets or lozenges formulated inconventional manner.

The optimized cytokine can be formulated for parenteral administrationby injection e.g., by bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form e.g., inampules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder-lyophilized form for constitutionwith a suitable vehicle, e.g., sterile pyrogen-free water, before use.

In addition to the formulations described previously, the optimizedcytokine also can be formulated as a depot preparation. Such long actingformulations can be administered by implantation (for example,subcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the therapeutic compounds can be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The active agents can be formulated for local or topical application,such as for topical application to the skin and mucous membranes, suchas in the eye, in the form of gels, creams, and lotions and forapplication to the eye or for intracisternal or intraspinal application.Such solutions, particularly those intended for ophthalmic use, can beformulated as 0.01%-10% isotonic solutions, pH about 5-7, withappropriate salts. The compounds can be formulated as aerosols fortopical application, such as by inhalation (see, e.g., U.S. Pat. Nos.4,044,126, 4,414,209, and 4,364,923, which describe aerosols fordelivery of a steroid useful for treatment inflammatory diseases,particularly asthma).

The concentration of active compound in the drug composition will dependon absorption, inactivation and excretion rates of the active compound,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art. For example, the amount that isdelivered is sufficient to treat the symptoms of hypertension.

The compositions, if desired, can be presented in a package, in kit or adispenser device, that can contain one or more unit dosage formscontaining the active ingredient. The package, for example, containsmetal or plastic foil, such as a blister pack. The pack or dispenserdevice can be accompanied by instructions for administration. Thecompositions containing the active agents can be packaged as articles ofmanufacture containing packaging material, an agent provided herein, anda label that indicates the disorder for which the agent is provided.

Methods of treatment of cytokine-mediated or cytokine-involved diseasesand immunotherapeutic methods are provided. The modified cytokines canbe used in any method of treatment for which the unmodified cytokine isused. Hence the modified cytokines can be used for treatment of alldisorders noted herein for the respective cytokines and for those knownto those of skill in the art for each of the others, such asimmunotherapeutic treatment (interleukins) and red blood cell expansionand stem cell expansion. The following table summarizes exemplary usesin addition to those noted herein of exemplary modified cytokinesprovided herein:

Cytokine Exemplary Uses, Diseases and Treatment IL-10 anti-inflammatorytreatment of chronic liver injury and disease; myeloma Interferon-gammaInterstitial/idiopathic pulmonary fibrosis; adjunctive immunotherapy forimmunosuppressed patients Granulocyte colony Crohn's disease; cardiacdisease; acquired and stimulating factor congenital neutropenias; asthmaLeukemia inhibitory myocardial infarction; multiple sclerosis; factorprevention of axonal atrophy; olfactory epithelium replacementstimulation Human growth growth hormone deficiency; acromegaly hormoneCiliary neurotrophic retinal degeneration treatments; factorneurodegenerative diseases such as Huntington's; auditory degenerativediseases Leptin obesity; pancreatitis; endometriosis Oncostatin Mchronic inflammatory diseases; rheumatoid arthritis; multiple sclerosis;tissue damage suppression Interleukin-6 Protection from liver injury;Crohn's disease; hematopoietic associated diseases Interleukin-12coxsackievirus treatment; neuroblastoma; melanoma, renal cell carcinoma;mucosal immunity induction Erythropoietin hypoxia; myocardial ischemia;anemia with renal failure and cancer treatments Granulocyte- stimulateantigen presenting cells; anti-tumor macrophage colony activity forleukemia, melanoma, and breast, liver stimulating factor and renal cellcarcinomas; adjunctive immunotherapy for immunosuppressed patients;autoimmune disease Interleukin-2 immune reactivation after chemotherapy;melanoma; colon carcinoma Interleukin-3 leukemia cell targeting; motorneuropathy; amyotrophic lateral sclerosis; asthma Interleukin-4 allergicasthma; lupus Interleukin-5 treatment for parasites; asthma; allergicdiseases accompanied by eosinophilia Interleukin-13 intracellularinfections; B-cell cancers; asthma Flt3 ligand prostate cancer; myeloidleukemia; engraftment of allogenic hematopoietic stem cells Stem cellfactor hepatic injury; asthma; hematopoietic engraftment

Treatment can be effected by any suitable route of administration usingsuitable formulations. If necessary, a particular dosage and durationand treatment protocol can be empirically determined or extrapolated.

K. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention. The specificmethods exemplified can be practiced with other species. The examplesare intended to exemplify generic processes.

Example 1

This example describes a plurality of chronological steps includingsteps from (i) to (viii):

-   -   (i) cloning of IFNα cDNA in a mammalian cell expression plasmid        (section A.1)    -   (ii) generation of a collection of targeted mutants on the IFNα        cDNA in the mammalian cell expression plasmid (section B)    -   (iii) production of IFNα mutants in mammalian cells (section        C.1)    -   (iv) screening and partial in vitro characterization of IFNα        mutants produced in mammalian cells in search of lead mutants        (section D)    -   (v) cloning of the lead mutants into a bacterial cell expression        plasmid (section A.2)    -   (vi) expression of lead mutants in bacterial cells (section C.2)    -   (vii) in vitro characterization of lead mutants produced in        bacteria (section D)    -   (viii) in vivo characterization of lead mutants produced in        bacteria (section E).        A. Cloning of IFNα-2b Encoding cDNA

A.1. Cloning of IFNα-2b cDNA in a Mammalian Cell Expression Plasmid

The IFNα-2b cDNA was first cloned into a mammalian expression vector,prior to the generation of the selected mutations. A collection ofmutants was then generated such that each individual mutant was createdand processed individually, physically separated from each other and inaddressable arrays. The mammalian expression vector pSSV9 CMV 0.3 pA wasengineered as follows:

The pSSV9 CMV 0.3 pA was cut by PvuII and religated (this step gets ridof the ITR functions), prior to the introduction of a new EcoRIrestriction site by Quickchange mutagenesis (Stratagene). Theoligonucleotides primers were:

EcoRI forward primer (SEQ ID NO: 218)5′-GCCTGTATGATTTATTGGATGTTGGAATTCCCTGATGCGGTATTTTC TCCTTACG-3′EcoRI reverse primer (SEQ ID NO: 219)5′-CGTAAGGAGAAAATACCGCATCAGGGAATTCCAACATCCAATAAATC ATACAGGC-3′.

The construct sequence was confirmed by using the followingoligonucleotides:

Seq ClaI forward primer: (SEQ ID NO: 220)5′-CTGATTATCAACCGGGGTACATATGATTGAC-ATGC-3′ Seq XmnI reverse primer(SEQ ID NO: 221) 5′-TACGGGATAATACCGCGCCACATAGCAGAA-C-3′.

Then, the XmnI-ClaI fragment containing the newly introduced EcoRI sitewas cloned into pSSV9 CMV 0.3 pA (SSV9 is a clone containing the entireadeno-associated virus (AAV) genome inserted into the PvuII site ofplasmid pEMBL (see, Du et al. (1996) Gene Ther 3:254-261)) to replacethe corresponding wild-type fragment and produce construct pSSV9-2EcoRI.

The DNA sequence of the IFNα-2b cDNA, which was inserted into themammalian vector pDG6 (ATCC accession No. 53169), was confirmed using apair of internal primers. The sequences of the IFNα-2b-relatedoligonucleotides for sequencing follow:

Seq forward primer: 5′-CCTGATGAAGGAGGACTC-3′ (SEQ ID NO: 222)Seq reverse primer: 5′-CCAAGCAGCAGATGAGTC-3′. (SEQ ID NO: 223)

Since the beginning of the IFNα-2b encoding cDNA (the signal peptideencoding sequence) is absent in pDG6, it was added using theoligonucleotide (see below) to the amplified gene. First, the IFNα-2bcDNA was amplified by PCR using pDG6 as template using the followingoligonucleotides as primers:

IFNα-2b 5′ primer (SEQ ID NO: 224) 5′-TCAGCTGCAAGTCAAGCTGCTCTGTGGGCTG-3′IFNα-2b 3′ primer (SEQ ID NO: 225)5′-GCTCTAGATCATTCCTTACTTCTTAAACTTTCTTGCAAGTTTGTTGA C-3′

The PCR product was then used in an overlapping PCR using the followingoligonucleotide sequences, having Hind III or XbaI restriction sites(underlined) or the DNA sequence missing in pDG6 (underlined):

IFNα-2b HindIII primer (SEQ ID NO: 226)5′-CCCAAGCTTATGGCCTTGACCTTTGCTTTACT-GGTG-3′ IFNα-2b XbaI primer(SEQ ID NO: 227) 5′-GCTCTAGATCATTCCTTACTTCTTAAACTTTCTTGCAAGTTTGTTGA C-3′IFNα-2b 80 bp 5′ primer (SEQ ID NO: 228) 5′-CCCAAGCTTATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAAGCTGCTCTGTGGGCTG-3′.

The entire IFNα-2b cDNA was cloned into the pTOPO-TA vector(Invitrogen). After checking gene sequence by automatic DNA sequencing;the HindIII-XbaI fragment containing the gene of interest was subclonedinto the corresponding sites of pSSV9-2EcoRI to producepAAV-EcoRI-IFNalpha-2b (pNB-AAV-IFN alpha-2b).

A.2 Cloning of the IFN α-2b Leads in an E. coli Expression Plasmid

A.2.1 Characterization of the Bacterial Cells

BL21-CodonPlus(DE3)-RP® competent Escherichia coli cells are derivedfrom Stratagene's high-performance BL21-Gold competent cells. Thesecells enable efficient high-level expression of heterologous proteins inE. coli. Efficient production of heterologous proteins in E. coli isfrequently limited by the rarity, in E. coli, of certain tRNAs that areabundant in the organisms from which the heterologous proteins arederived. Availability of tRNAs allows high-level expression of manyheterologous recombinant genes in BL21-Codon Plus cells that are poorlyexpressed in conventional BL21 strains. BL21-CodonPLus(DE3)-RP cellscontain a ColE1-compatible, pACYC-based plasmid containing extra copiesof the argU and proL tRNA genes.

A.2.2 Cloning of Wild-Type IFN α

To express IFNα-2b in E. coli cDNA encoding the mature form of IFNα-2bwas finally cloned into the plasmid pET-11 (Novagen). Briefly, this cDNAfragment was amplified by PCR using the primers SEQ ID Nos. 1306 and1305, respectively:

FOR-IFNA- 5′ AACATATGTGTGATCTGCCTCAAACCCACAGCCTGGG TAGC 3′ REV-IFNA- 5′AAGGATCCTCATTCCTTACTTCTTAAACTTTCTTGCA AGTTTGTTG 3′,from pSSV9-EcoRI-IFN α-2b (see above), which contains full-length IFN-2alpha cDNA as a matrix, using Herculase DNA-polymerase (Stratagene). ThePCR fragment was subcloned into pTOPO-TA vector (Invitrogen) yieldingpTOPO-IFN α-2b. The sequence was verified by sequencing. pET11 IFN α-2bwas prepared by insertion of the NdeI-Bam HI (Biolabs) fragment frompTOPO-IFN α-2b into the NdeI-Bam HI sites of pET 11. The DNA sequence ofthe resulting pET 11-IFN α-2b construct was verified by sequencing andthe plasmid was used for IFN α-2b expression in E. coli.

A.2.3 Cloning of IFN α-2b Mutants from the Mammalian Expression Plasmidinto the E. coli Expression Plasmid

Lead mutants of Interferon alpha were first generated in thepSSV9-IFNα-EcoRI plasmid. With the only exception of E159H and E159Q,all mutants were amplified using the primers below. Primers containedNdeI (in Forward) and BamHI (in Reverse) restriction sites:

FOR-IFNA- (SEQ ID No. 1306 5′ AACATATGTGTGATCTGCCTCAAACCCACAGCCTGGGTAGC 3′; and REV-IFNA- (SEQ ID No. 1305) 5′AAGGATCCTCATTCCTTACTTCTTAAACTTTCTTGC AAGTTTGTTG 3′.Mutants E159H and E159Q were amplified using the following primers onreverse side (primer forward was the same than described above):

REV-IFNA-E159H- SEQ ID No. 1304 5′AAGGATCCTCATTCCTTACTTCTTAAACTGTGTTGCAAGT TTGTTG 3′ above; andREV-IFNA-E159Q- SEQ ID No. 1305 5′AAGGATCCTCATTCCTTACTTCTTAAACTCTGTTGCAAGT TTGTTG 3′.Mutants were amplified with Pfu Turbo Polymerase (Stratagene). PCRproducts were cloned into pTOPO plasmid (Zero Blunt TOPO PCR cloningkit, Invitrogen). The presence of the desired mutations was checked byautomatic sequencing. The NdeI+BamHI fragment of the pTOPO-IFNα positiveclones was then cloned into NdeI+BamHI sites of the pET11 plasmid.B. Construction of a Collection of IFNα-2b Mutants in a MammalianExpression Plasmid

A series of mutagenic primers was designed to generate the appropriatesite-specific mutations in the IFNα-2b cDNA. Mutagenesis reactions wereperformed with the Chameleon mutagenesis kit (Stratagene) usingpNB-AAV-IFNα-2b as the template. Each individual mutagenesis reactionwas designed to generate one single mutant protein. Each individualmutagenesis reaction contains one and only one mutagenic primer. Foreach reaction, 25 pmoles of each (phosphorylated) mutagenic primer weremixed with 0.25 pmoles of template, 25 pmoles of selection primer(introducing a new restriction site), and 2 μl of 10× mutagenesis buffer(100 mM Tris-acetate pH 7.5; 100 mM MgOAc; 500 mM KOAc pH 7.5) into eachwell of 96 well-plates. To allow DNA annealing, PCR plates wereincubated at 98° C. during 5 min and immediately placed 5 min on ice,before incubating at room temperature during 30 min. Elongation andligation reactions were allowed by addition of 7 μl of nucleotide mix(2.86 mM each nucleotide; 1.43× mutagenesis buffer) and 3 μl of afreshly prepared enzyme mixture of dilution buffer (20 mM Tris HClpH7.5; 10 mM KCl; 10 mM β-mercaptoethanol; 1 mM DTT; 0.1 mM EDTA; 50%glycerol), native T7 DNA polymerase (0.025 U/μl), and T4 DNA ligase (1U/μl) in a ratio of 1:10, respectively. Reactions were incubated at 37°C. for 1 h before inactivation of T4 DNA ligase at 72° C. during 15 min.In order to eliminate the parental plasmid, 30 μl of a mixturecontaining 1× enzyme buffer and 10 U of restriction enzyme was added tothe mutagenic reactions followed by incubation at 37° C. for at least 3hours. Next, 90 μl aliquots of XLmutS competent cells (Stratagene)containing 25 mM β-mercaptoethanol were place in ice-chilled deep-wellplates. Then, plates were incubated on ice for 10 min with gentle vortexevery 2 min. Transformation of competent cells was performed by addingaliquots of the restriction reactions ( 1/10 of reaction volume) andincubating on ice for 30 min. A heat pulse was performed in a 42° C.water bath for 45 s, followed by incubation on ice for 2 minutes.Preheated SOC medium (0.45 ml) was added to each well and plates wereincubated at 37° C. for 1 h with shaking. In order to enrich for mutatedplasmids, 1 ml of 2×YT broth medium supplemented with 100 μg/mlampicillin was added to each transformation mixture followed byovernight incubation at 37° C. with shaking. Plasmid DNA isolation wasperformed by alkaline lysis using Nucleospin Multi-96 Plus Plasmid Kit(Macherey-Nagel) according to the manufacturer's instructions. Selectionof mutated plasmids was performed by digesting 500 μg of plasmidpreparation with 10 U of selection endonuclease in an overnightincubation at 37° C. A fraction of the digested reactions ( 1/10 of thetotal volume) was transformed into 40 μl of Epicurian coli XL1-Bluecompetent cells (Stratagene) supplemented with 25 mM β-mercaptoethanol.

Transformation was performed was as described above. Transformants wereselected on LB-ampicillin agar plates incubated overnight at 37° C.Isolated colonies were picked up and grown overnight at 37° C. intodeep-well plates. Four clones per reaction were screened by endonucleasedigestion of a new restriction site introduced by the selection primer.Finally, each mutation that was introduced to produce this collection ofcandidate LEAD IFNα-2b mutant plasmids encoding the proteins set forthin Table 2 of Example 2 was confirmed by automatic DNA sequencing.

C. Production of IFNα-2b Mutants

C.1 In Mammalian Cells

IFNα-2b mutants were produced in 293 human embryo kidney (HEK) cells(obtained from ATCC), using Dulbecco's modified Eagle's mediumsupplemented with glucose (4.5 g/L; Gibco-BRL) and fetal bovine serum(10%, Hyclone). Cells were transiently transfected with the plasmidsencoding the IFNα-2b mutants as follows: 0.6×10⁵ cells were seeded into6 well-plates and grown for 36 h before transfection. Confluent cells atabout 70%, were supplemented with 2.5 μg of plasmid (IFNα-2b mutants)and 10 mM poly-ethylene-imine (25 KDa PEI, Sigma-Aldrich). After gentlyshaking, cells were incubated for 16 h. Then, the culture medium waschanged with 1 ml of fresh medium supplemented with 1% of serum. IFNα-2bwas measured on culture supernatants obtained 40 h after transfectionand stored in aliquots at −80° C. until use.

Supernatants containing IFNα-2b from transfected cells were screenedfollowing sequential biological assays as follows. Normalization ofIFNα-2b concentration from culture supernatants was performed byenzyme-linked immunoabsorbent assay (ELISA) using a commercial kit (R &D) and following the manufacturer's instructions. This assay includesplates coated with an IFNα-2b monoclonal antibody that can be developedby coupling a secondary antibody conjugated to the horseradishperoxidase (HRP). IFNα-2b concentrations on samples containing (i) wildtype IFNα-2b produced under comparable conditions as the mutants, (ii)the IFNα-2b mutants and (iii) control samples (produced from cellsexpressing GFP) were estimated by using an international referencestandard provided by the NIBSC, UK.

C.2 In Bacteria

A volume of 200 ml of culture medium (LB/Ampicillin/Chloramphenicol) wasinoculated with 5 ml of pre-culture BL21-pCodon+-pET-IFN α-2b mutaovernight at 37° C. with constant shaking (225 rpm). The production ofIFN α-2b was induced by the addition of 50 μl of 2M IPTG atDO_(600nm)˜0.6.

The culture was continued for 3 additional hours and was centrifuged at4° C. and 5000 g for 15 minutes. The supernatant (culture medium) wasdiscarded and bacteria were lysed in 8 ml of lysis buffer by thermalshock (freezing-thawing: 37° C.-15 min; −80° C.-10 min; 37° C.-15 min;−80° C.-10 min; 37° C.-15 min). After centrifugation (10000 g, 15 min,4° C.), the supernatant (soluble proteins fraction) was discarded, andthe precipitated material (insoluble protein fraction containing the IFNα-2b protein as inclusion bodies) was purified.

C.3 Pre-purification of IFN α-2b as Inclusion Bodies in E. coli

C.3.1 Washing of Inclusion Bodies by Sonication

Pellets containing the inclusion bodies were suspended in 10 ml ofbuffer and sonicated (80 watts) on ice, 1 second “on,” 1 second “off”for a total of 4 min. Suspensions were then centrifuged (4° C., 10000 g,15 min), and supernatants were recovered. Pellets were resuspended in 10ml of buffer for a new sonication/centrifugation cycle. Triton X-100 wasthen eliminated by two additional cycles of sonication/centrifugationwith buffer. Pellets containing the inclusion bodies were recovered anddissolved. The washed supernatants were stored at 4° C.

C.3.2 Solubilization of Inclusion Bodies by Denaturation

Once washed, the inclusion bodies were solubilized in buffer at aconcentration estimated in 0.3 mg/ml measuring the OD280 (consideringthe coefficient of molar extinction of IFN α-2b). Solubilization wascarried out overnight at 4° C., under shaking.

C.3.3 Renaturation of IFN α-2b by Dialysis of GdnHCl

Samples contained 1 mg of protein at 0.3 mg/ml (5 ml in total) inbuffer. The GdnHCl (Guanidium hydrochloride) present in the samples waseliminated by dialysis (minimum membrane cut=10 kDa) overnight at 4° C.against buffer (1 litre) (final concentration of GdnHCl: 43 mM). Next,samples were further dialysed against 1 litre of buffer during 2.5 h.This step was repeated two additional times. After dialysis, very littleprecipitate was visible.

D. Screening and In Vitro Characterization of IFN α-2b Mutants

Two activities were measured directly on IFN samples: antiviral andantiproliferation activities. Dose (concentration)-response (activity)experiments for antiviral or antiproliferation activity permittedcalculation of the “potency” for antiviral and antiproliferationactivities, respectively. Antiviral and antiproliferation activitiesalso were measured after incubation with proteolytic samples, such asspecific proteases, mixtures of selected proteases, human serum or humanblood. Assessment of activity following incubation with proteolyticsamples allowed to determine the residual (antiviral orantiproliferation) activity and the respective kinetics of half-lifeupon exposure to proteases.

D.1. Antiviral Activity

IFNα-2b protects cells against viral infection by a complex mechanismdevoted to create an unfavorable environment for viral proliferation.Cellular antiviral response due to IFNα-2b (IFN anti-viral assay) wasassessed using an interferon-sensitive HeLa cell line (ATCC accessionno. CCL-2) treated with the encephalomyocarditis virus (EMCV). Theassessment of either the virus-induced cytopathic effects (CPE) or theamount of EMCV mRNA in extracts of infected cells by RT-PCR was used todetermine IFNα activity in samples.

D.1.1 Antiviral Activity—Measure by RT-PCR

Confluent cells were trypsinized and plated at density 2×10⁴ cells/wellin DMEM 5% SVF medium (Day 0). Cells were incubated with IFN α-2b (at aconcentration of 500 U/ml) to get 500 pg/ml and 150 pg/well (100 μl ofIFN solution), during 24 h at 37° C. prior to be challenged with EMCV(1/1000 dilution; MOI 100). After an incubation of 16 h, whenvirus-induced CPE was near maximum in untreated cells, the number ofEMCV particles in each well was determined by RT-PCR quantification ofEMCV mRNA, using lysates of infected cells. RNA from cell extracts waspurified after a DNAse/proteinase K treatment (Applied Biosystems). TheCPE was evaluated using both Uptibleu (Interchim) and MTS (Promega)methods, which are based on detecting bio-reductions produced by themetabolic activity of cells in a fluorometric and colorimetric manner,respectively. In order to produce a standard curve for EMCVquantification, a 22 bp DNA fragment of the capsid protein-cDNA wasamplified by PCR and cloned into pTOPO-TA vector (Invitrogen). Next,RT-PCR quantification of known amounts of pTOPO-TA-EMCV capsid gene wasperformed using the One-step RT-PCR kit (Applied Biosystems) and thefollowing EMCV-related (cloning) oligonucleotides and probe:

EMCV forward primer (SEQ ID NO: 229) 5′-CCCCTACATTGAGGCATCCA-3′EMCV reverse primer (SEQ ID NO: 230) 5′-CAGGAGCAGGACAAGGTCACT-3′EMCV probe (SEQ ID NO: 231) 5′-(FAM)CAGCCGTCAAGACCCAACCGCT(TAMR A)-3′.

D.1.2 Antiviral Activity—Measure by CPE

Antiviral activity of IFN α-2b was determined by the capacity of thecytokine to protect HeLa cells against EMC (mouse encephalomyocarditis)virus-induced cytopathic effects. The day before, HeLa cells (2×10⁵cells/ml) were seeded in flat-bottomed 96-well plates containing 100μl/well of Dulbecco's MEM-GlutamaxI-sodium pyruvate medium supplementedwith 5% SVF and 0.2% of gentamicin. Cells were growth at 37° C. in anatmosphere of 5% CO₂ for 24 hours.

Two-fold serial dilutions of interferon samples were made with MEMcomplete media into 96-Deep-Well plates with final concentration rangingfrom 1600 to 0.6 pg/ml. The medium was aspirated from each well and 100μl of interferon dilutions were added to HeLa cells. Each interferonsample dilution was assessed in triplicate. The two last rows of theplates were filled with 100 μl of medium without interferon dilutionsamples in order to serve as controls for cells with and without virus.

After 24 hours of growth, a 1/1000 EMC virus dilution solution wasplaced in each well except for the cell control row. Plates werereturned to the CO₂ incubator for 48 hours. Then, the medium wasaspirated and the cells were stained for 1 hour with 100 μl of Bluestaining solution to determine the proportion of intact cells. Plateswere washed in a distilled water bath. The cell bound dye was extractedusing 100 μl of ethylene-glycol mono-ethyl-ether (Sigma). The absorbanceof the dye was measured using an Elisa plate reader (Spectramax). Theantiviral activity of IFN α-2b samples (expressed as number of IU/mg ofproteins) was determined as the concentration needed for 50% protectionof the cells against EMC virus-induced cytopathic effects. Forproteolysis experiments, each point of for the kinetic measurements wasassessed at 500 and 166 pg/ml in triplicate.

D.2 Antiproliferation Activity

Anti-proliferative activity of interferon α-2b was determined by thecapacity of the cytokine to inhibit proliferation of Daudi cells. Daudicells (1×10⁴ cells) were seeded in flat-bottomed 96-well platescontaining 50 μl/well of RPMI 1640 medium supplemented with 10% SVF, 1×glutamine and 1 ml of gentamicin. No cell was added to the last row (“H”row) of the flat-bottomed 96-well plates in order to evaluate backgroundabsorbance of culture medium.

At the same time, two-fold serial dilutions of interferon samples weremade with RPMI 1640 complete media into 96-Deep-Well plates with finalconcentration ranging from 6000 to 2.9 pg/ml. Interferon dilutions (50μl) were added to each well containing 50 μl of Daudi cells. The totalvolume in each well should now be 100 μl. Each interferon sampledilution was assessed in triplicate. Each well of the “G” row of theplates was filled with 50 μl of RPMI 1640 complete media in order to beused as positive control. The plates are incubated for 72 hours at 37°C. in a humidified, 5% CO₂ atmosphere.

After 72 hours of growth, 20 μl of Cell titer 96 Aqueous one solutionreagent (Promega) was added to each well and incubated 1H30 at 37° C. inan atmosphere of 5% CO₂. To measure the amount of colored solubleformazan produced by cellular reduction of the MTS, the absorbance ofthe dye was measured using an Elisa plate reader (Spectramax) at 490 nm.

The corrected absorbances (“H” row background value subtracted) obtainedat 490 nm were plotted versus concentration of cytokine. The ED50 valuewas calculated by determining the X-axis value corresponding to one-halfthe difference between the maximum and minimum absorbance values.(ED50=the concentration of cytokine necessary to give one-half themaximum response).

D.3 Treatment of IFN α-2b with Proteolytic Preparations

Mutants were treated with proteases in order to identify resistantmolecules. The resistance of the mutant IFN α-2b molecules compared towild-type IFN α-2b against enzymatic cleavage (30 min, 25° C.) by amixture of proteases (containing 1.5 μg of each of the followingproteases (1% wt/wt, Sigma): α-chymotrypsin, carboxypeptidase,endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C,endoproteinase Lys-C, and trypsin) was determined. At the end of theincubation time, 10 μl of anti-proteases complete, mini EDTA free, Roche(one tablet was dissolved in 10 ml of DMEM and then diluted to 1/1000)was added to each reaction in order to inhibit protease activity.Treated samples were then used to determine residual antiviral orantiproliferation activities.

D.4 Protease Resistance—Kinetic Analysis

The percent of residual IFN α-2b activity over time of exposure toproteases was evaluated by a kinetic study using either (a) 15 pg ofchymotrypsin (10% wt/wt), (b) a lysate of human blood at dilution 1/100,(c) 1.5 pg of protease mixture, or (d) human serum. Incubation timeswere: 0 h, 0.5 h, 1 h, 4 h, 8 h, 16 h, 24 h and 48 h. Briefly, 20 μl ofeach proteolytic sample (proteases, serum, blood) was added to 100 μl ofIFN α2b at 1500 pg/ml (500 U/ml) and incubated for variable times, asindicated. At the appropriate time points, 10 μl of anti-proteasesmixture, mini EDTA free, Roche (one tablet was dissolved in 10 ml ofDMEM and then diluted to 1/500) was added to each well in order to stopproteolysis reactions. Biological activity assays were then performed asdescribed for each sample in order to determine the residual activity ateach time point.

D.5 Performance

The various biological activities, protease resistance and potency ofeach individual mutant were analyzed using a mathematical model andalgorithm (NautScan™; described in French Patent No. 9915884; (publishedas International PCT application No. WO 01/44809 based on PCT noPCT/FR00/03503). Data was processed using a Hill equation-based modelthat uses key feature indicators of the performance of each individualmutant. Mutants were ranked based on the values of their individualperformance and those on the top of the ranking list were selected asleads.

E. Pharmacokinetics of Selected Lead Mutants in Mice

IFNα-2b mutants selected on the basis of their overall performance invitro, were tested for pharmacokinetics in mice in order to have anindication of their half-life in blood in vivo. Mice were treated bysubcutaneous (SC) injection with aliquots of each of a number ofselected lead mutants. Blood was collected at increasing time pointsbetween 0.5 and 48 hours after injection. Immediately after collection,20 ml of anti-protease solution were added to each blood sample. Serumwas obtained for further analysis. Residual IFN-α activity in blood wasdetermined using the tests described in the precedent sections for invitro characterization. Wild-type IFN α (that had been produced inbacteria under comparable conditions as the lead mutants) as well as apegylated derivative of IFN α, Pegasys (Roche), also were tested forpharmacokinetics in the same experiments.

Example 2

This example demonstrates the 2-dimensional (2D) scanning of IFNα-2b forincreased resistance to proteolysis. For results, see FIGS. 6(A)-6(N),6(T) and 6(U).

A) Identifying Some or all Possible Target Sites on the Protein Sequencethat are Susceptible to Digestion by One or More Specific Proteases(these Sites are the is-HITs).

Because IFNα-2b is administered as a therapeutic protein in the bloodstream, a set of proteases was identified that were expected to broadlymimic the protease contents in serum. From that list of proteases, alist of the corresponding target amino acids was identified (shown inparenthesis) as follows: α-chymotrypsin (F, L, M, W, and Y),endoproteinase Arg-C(R), endoproteinase Asp-N (D), endoproteinase Glu-C(E), endoproteinase Lys-C (K), and trypsin (K and R) Carboxypeptidase Y,which cleaves non-specifically from the carboxy-terminal ends ofproteins, was also included in the protease mixture. The distribution ofthe target amino acids over the protein sequence spreads over thecomplete length of the protein, suggesting that the protein ispotentially sensitive to protease digestion all over its sequence (FIG.1A). In order to restrict the number of is-HITs to a lower number ofcandidate positions, the 3-dimensional structure of the IFNα-2b molecule(PDB code 1RH2) was used to identify and select only those residuesexposed on the surface, while discarding from the candidate list thosewhich remain buried in the structure, and therefore stay lesssusceptible to proteolysis (FIG. 1B).

B) Identifying Appropriate Replacing Amino Acids, Specific for Eachis-HIT, Such That if Used to Replace One or More of the Original, Suchas Native, Amino Acids at That Specific is-HIT, They can be Expected toIncrease the is-HIT Amino Acid Position's Resistance to Digestion byProtease While at the Same Time, Maintaining or Improving the RequisiteBiological Activity of the Protein (These Replacing Amino Acids are theCandidate LEADs).

To select the candidate replacing amino acids for each is-HIT position,PAM250 matrix based analysis was used (FIG. 2). In one embodiment, thetwo highest values in PAM250 matrix, corresponding to the highestoccurrence of substitutions between residues (“conservativesubstitutions” or “accepted point mutations”), were chosen (FIG. 3).Whenever only a conservative substitution was available for a given highvalue of the PAM250, the following higher value was selected and thetotality of conservative substitutions for this value was considered.The replacement of amino acids that are exposed on the surface bycysteine residues (as shown in FIG. 3, while replacing Y by H or I) wasexplicitly avoided, since this change would potentially lead to theformation of intermolecular disulfide bonds.

Thus, based on the nature of the challenging proteases, and onevolutionary considerations as well as protein structural analysis, astrategy was defined for the rational design of human IFNα-2b mutantshaving increased resistance to proteolysis which could producetherapeutic proteins having a longer half-life. By using the algorithmPROTEOL (see, e.g., infobiogen.fr), a list of residues along the IFNα-2bsequence was established, which can be recognized as a substrate fordifferent enzymes present in the serum. Because the number of residuesin this particular list was high, the 3-dimensional structure of IFNα-2bobtained from the NMR structure of IFNα-2a (PDB code 1ITF) was used toselect only those residues exposed to the solvent. Using this approach,42 positions were identified, which numbering is that of the matureprotein (SEQ ID NO:1): L3, P4, R12, R13, M16, R22, R23, F27, L30, K31,R33, E41, K49, E58, K70, E78, K83, Y89, E96, E107, P109, L110, M111,E113, L117, R120, K121, R125, L128, K131, E132, K133, K134, Y135, P137,M148, R149, E159, L161, R162, K164, and E165. Each of these positionswas replaced by amino acid residues, such that they are defined ascompatible by the substitution matrix PAM250 while at the same time thereplacement amino acids do not generate new sites for proteases.

The list of performed residue substitutions as determined by PAM250analysis is as follows:

R to H, Q

E to H, Q

K to Q, T

L to V, I

M to I, V

P to A, S

Y to I, H.

C) Systematically Introducing the Specific Replacing Amino Acids(Candidate LEADs) at Every Specific is-HIT Position to Generate aCollection Containing the Corresponding Mutant Molecules.

The individual IFNα-2b mutants are generated, produced andphenotypically characterized one-by-one, in addressable arrays as setforth in Example 1, such that each mutant molecule contains initiallyamino acid replacements at only one is-HIT site. LEAD positions wereobtained in IFNα-2b variants after a screening for protection againstproteases, and comparing protease-untreated and protease-treated variantpreparations with the corresponding conditions for the wild-typeIFNα-2b. The percent of residual (anti-viral) activity for the IFNα-2bE113H variant after treatment with chymotrypsin, protease mixture, bloodlysate or serum was compared to the treated wild-type IFNα-2b. SelectedIFNα-2b LEADs are shown in Table 2.

A top and side view of IFNα-2b structure in ribbon representation(obtained from NMR structure of IFNα-2a, PDB code 1ITF) depict residuesin “space filling” defining (1) the “receptor binding region” as deducedeither by “alanine scanning” data and studies by Piehler et al., J.Biol. Chem., 275:40425-40433, 2000, and Roisman et al., Proc. Natl.Acad. Sci USA, 98:13231-13236, 2001, and (2) replacing residues (LEADs)for resistance to proteolysis.

TABLE 2 Selected LEADs of IFNα-2b following protease resistance SEQ IDProteolysis IFN antiviral Mutant No. protection activity F27V 83 Pseudowt Pseudo wt R33H 86 Pseudo wt Pseudo wt E41Q 87 Increased IncreasedE41H 88 Pseudo wt Increased E58Q 89 Increased Pseudo wt E58H 90Increased Increased E78Q 92 Increased Increased E78H 93 IncreasedIncreased Y89H 1303 Pseudo wt Pseudo wt E107Q 95 Increased Pseudo wtE107H 96 Increased Pseudo wt P109A 97 Pseudo wt Pseudo wt L110V 98Pseudo wt Pseudo wt M111V 978 Pseudo wt Pseudo wt E113H 101 IncreasedPseudo wt L117V 102 Increased Pseudo wt L117I 103 Increased Pseudo wtK121Q 104 Increased Pseudo wt R125H 106 Increased Increased R125Q 107Increased Increased K133Q 114 Increased Increased E159H 125 IncreasedPseudo wt E159Q 124 Increased Pseudo wt

Example 3 Stabilization of IFNα-2b by Creation of N-Glycosylation Sites

The creation of N-glycosylation sites on the protein was a secondstrategy that was used to stabilize IFNα-2b. Natural human IFNα-2bcontains a unique O-glycosylation site at position 129 (the numberingcorresponds to the mature protein; SEQ ID NO:1), however, noN-glycosylation sites are found in this sequence. N-glycosylation sitesare defined by the N-X-S or N-X-T consensus sequences. Glycosylation hasbeen found to play a role in protein stability. For example,glycosylation has been found to increase bioavailability via highermetabolic stability and reduced clearance. In order to generate morestable IFNα-2b variants, the N-glycosylation consensus sequencesindicated above were introduced in the IFNα-2b sequence by mutagenesis.Variants of IFNα-2b carrying new glycosylation sites were assessed aspreviously described.

The structure of IFNα-2b is characterized by a helix bundle composed of5 helices (A, B, C, D and E) connected with each other by a series ofloops (a large AB loop and three shorter BC, CD, DE loops). The helicesare joined together by two disulfide bridges between residues 1/98 and29/138 of SEQ ID NO:1. The loops are contemplated herein to representpreferential sites for glycosylation given their exposure. Therefore,N-glycosylation sites (N-X-S or N-X-T) were created in each of the loopsequences (Table 3). Selected LEADs and pseudo wild-type IFNα-2b mutantsafter screening for addition of glycosylation sites are shown in Table4.

TABLE 3 In silico HITs for addition of glycosylation sites on IFNα-2bCodon No. SEQ ID No. N-X-S SEQ ID No. N-X-T c2-4 D2N/P4S D2N/P4T c3-5L3N/Q5S L3N/Q5T c4-6 P4N/T6S P4N/T6T c5-7 127 Q5N/H7S 128 Q5N/H7T c6-8129 T6N/S8S T6N/S8T c7-9 H7N/L9S H7N/L9T c8-10 130 S8N/G10S 131 S8N/G10Tc9-11 L9N/S11S L9N/S11T c10-12 132 M21N/R23S M21N/R23T c22-24 R22N/I24SR22N/I24T c23-25 R23N/S25S 133 R23N/S25T c24-26 134 I24N/L26S I24N/L26Tc25-27 135 S25N/F27S 136 S25N/F27T c26-28 137 L26N/S28S 138 L26N/S28Tc28-30 S28N/L30S S28N/L30T c30-32 139 L30N/D32S L30N/D32T c31-33K31N/R33S K31N/R33T c32-34 D32N/H34S D32N/H34T c33-35 140 R33N/D35S 141R33N/D35T c34-36 142 H34N/F36S 143 H34N/F36T c35-37 144 D35N/G37SD35N/G37T c36-38 145 F36N/F38S 146 F36N/F38T c37-39 G37N/P39S 147G37N/P39T c38-40 148 F38N/Q40S 149 F38N/Q40T c39-41 150 P39N/E41S 151P39N/E41T c40-42 152 Q40N/E42S 153 Q40N/E42T c41-43 E41N/F43S 155E41N/F43T c42-44 E42N/G44S E42N/G44T c43-45 F43N/N45S F43N/N45T c44-46156 G44N/Q46S 157 G44N/Q46T c45-47 158 N45N/F47S 159 N45N/F47T c46-48160 Q46N/Q48S 161 Q46N/Q48T c47-49 162 F47N/K49S 163 F47N/K49T c48-50Q48N/A50S Q48N/A50T c49-51 164 K49N/E51S K49N/E51T c50-52 A50N/T52SA50N/T52T c68-70 S68N/K70S S68N/K70T c70-72 K70N/S72S K70N/S72T c75-77165 A75N/D77S A75N/D77T c77-79 D77N/T79S D77N/T79T C100-102 166I100N/G102S 167 I100N/G102T C101-103 Q101N/V103S Q101N/V103T C102-104G102N/G104S G102N/G104T C103-105 168 V103N/V105S 169 V103N/V105TC104-106 G104N/T106S 170 G104N/T106T C105-107 171 V105N/E107SV105N/E107T C10--108 172 T106N/T108S 173 T106N/T108T C107-109 174E107N/P109S 175 E107N/P109T C108-110 T108N/I110S T108N/I110T C134-136K134N/S136S 176 K134N/S136T C154-156 S154N/N156S S154N/N156T C155-157T155N/L157S T155N/L157T C156-158 N156N/Q158S N156N/Q158T C157-159 177L157N/E159S 178 L157N/E159T C158-160 Q158N/S160S 179 Q158N/S160TC159-161 180 E159N/L161S 181 E159N/L161T C160-162 S160N/R162SS160N/R162T C161-163 L161N/S163S L161N/S163T C162-164 R162N/K164SR162N/K164T C163-165 S163N/E165S S163N/E165T

TABLE 4 Selected LEADs and pseudo wild-type IFNα-2b mutants afterscreening for addition of glycosylation sites Proteolysis IFN antiviralMutant SEQ ID No. protection activity Q5N/H7S 127 Increased Pseudo wtQ5N/H7T 128 ND* ND P39N/E41S 150 Increased Pseudo wt P39N/E41T 151Increased Pseudo wt Q40N/E42S 152 Increased Pseudo wt Q40N/E42T 153Increased Pseudo wt E41N/F43S 154 Increased Pseudo wt E41N/F43T 155Increased Pseudo wt F43N/N45S Increased Pseudo wt F43N/N45T ND NDG44N/Q46S 156 ND ND G44N/Q46T 157 Increased Pseudo wt N45N/F47S 158Increased Pseudo wt N45N/F47T 159 Increased Pseudo wt Q46N/Q48S 160Increased Pseudo wt Q46N/Q48T 161 ND ND F47N/K49S 162 Increased Pseudowt F47N/K49T 163 Increased Pseudo wt I100N/G102S 166 Pseudo wt IncreasedI100N/G102T 167 Pseudo wt Increased V105N/E107S 171 Pseudo wt IncreasedV105N/E107T Pseudo wt Increased T106N/T108S 172 Pseudo wt IncreasedT106N/T108T 173 Pseudo wt Increased E107N/P109S 174 Pseudo wt IncreasedE107N/P109T 175 Pseudo wt Increased L157N/E159S 177 Pseudo wt IncreasedL157N/E159T 178 Pseudo wt Increased E159N/L161S 180 Pseudo wt IncreasedE159N/L161T 181 Pseudo wt Increased *ND, not determined

Example 4 Redesign of Interferon α-2b Proteins

The use of the protein redesign approach provided herein permits thegeneration of proteins such that they maintain requisite levels andtypes of biological activity compared to the native protein while theirunderlying amino acid sequences have been significantly changed by aminoacid replacement. To first identify those amino acid positions on theIFNα-2b protein that are involved or not involved IFNα-2b proteinactivity, such as binding activity of IFNα-2b to its receptor, anAla-scan was performed on the IFNα-2b sequence. For this purpose, eachamino acid in the IFNα-2b protein sequence was individually changed intoAlanine. Any other amino acid, particularly another amino acid that hasa neutral effect on structure, such as Gly or Ser, also can be used.Each resulting mutant IFNα-2b protein was then expressed and theantiviral activity of the individual mutants was assayed. The particularamino acid positions that are sensitive to replacement by Ala, referredto herein as HITs would in principle not be suitable targets for aminoacid replacement to increase protein stability, because of theirinvolvement in the activity of the molecule. For the Ala-scanning, thebiological activity measured for the IFNα-2b molecules was: i) theircapacity to inhibit virus replication when added to permissive cellspreviously infected with the appropriate virus and, ii) their capacityto stimulate cell proliferation when added to the appropriate cells. Therelative activity of each individual mutant compared to the nativeprotein was assayed. HITS are those mutants that produce a decrease inthe activity of the protein (e.g., in this example, all the mutants withactivities below about 30% of the native activity).

In addition, to identify the HIT positions, the Alanine-scan was used toidentify the amino acid residues on IFNα-2b that when replaced withalanine lead to a ‘pseudo-wild type’ activity, i.e., those that can bereplaced by alanine without leading to a decrease in biologicalactivity.

A collection of mutant molecules was generated and phenotypicallycharacterized such that IFNα-2b proteins with amino acid sequencesdifferent from the native ones but that still elicit the same level andtype of activity as the native protein were selected. HITs and pseudowild-type amino acid positions are shown in Table 5.

TABLE 5 HITs and pseudo wild-type positions to IFNα-2b redesign HITs(viral Pseudo wt (viral Mutants SEQ ID No. activity) activity) D2A 2Decreased P4A 3 Pseudo wt Q5A 4 Pseudo wt T6A 5 Pseudo wt H7A 6Decreased S8A 7 Decreased L9A 8 Pseudo wt G10A 9 Pseudo wt S11A 10Decreased R12A 11 Decreased R13A 12 Decreased T14A 13 Decreased L15A 14Decreased M16A 15 Decreased L17A 16 Pseudo wt Q20A 17 Pseudo wt R23A 18Decreased I24A 19 Pseudo wt S25A 20 Pseudo wt L26A 21 Decreased S28A 22Decreased C29A 23 Decreased L30A 24 Decreased K31A 25 Decreased D32A 26Decreased R33A 27 Decreased D35A 28 Pseudo wt G37A 29 Pseudo wt G39A 30Pseudo wt E41A 31 Pseudo wt E42A 32 Pseudo wt F43A 33 Decreased N45A 34Decreased F47A 35 Decreased E51A 36 Pseudo wt T52A 37 Pseudo wt I53A 38Decreased P54A 39 Pseudo wt V55A 40 Pseudo wt L56A 41 Pseudo wt H57A 42Pseudo wt E58A 43 Pseudo wt M59A 44 Decreased I60A 45 Pseudo wt I63A 46Pseudo wt F64A 47 Pseudo wt N65A 48 Pseudo wt L66A 49 Decreased F67A 50Decreased T69A 51 Decreased K70A 52 Decreased D71A 53 Decreased S72A 54Decreased W76A 55 Pseudo wt D77A 56 Pseudo wt E78A 57 Pseudo wt L81A 58Pseudo wt D82A 59 Decreased K83A 60 Decreased F84A 61 Decreased Y85A 62Pseudo wt Y89A 63 Pseudo wt Q90A 64 Pseudo wt Q91A 65 Decreased N93A 66Decreased D94A 67 Decreased C98A 68 Decreased V99A 69 Decreased Q101ADecreased G104A 70 Pseudo wt L110A 71 Pseudo wt S115A 72 Pseudo wt Y122A73 Decreased W140A 74 Decreased E146A 75 Pseudo wt

Example 5 Super LEADS of Interferon α-2b Protein by Additive DirectionalMutagenesis

The use of an additive directional mutagenesis approach provided amethod for the assembly of multiple mutations previously present on theindividual LEAD molecules in a single mutant protein thereby generatingsuper-LEAD mutant proteins. In this method, a collection of nucleic acidmolecules encoding a library of new mutant molecules is generated,tested and phenotypically characterized one-by-one in addressablearrays. Super-LEAD mutant molecules are such that each molecule containsa variable number and type of LEAD mutations

Using the LEADs obtained in Example 2, six series of mutant moleculeswere generated with more than one mutation per molecule as shown inTable 6. Some SuperLEAD mutant molecules were phenotypicallycharacterized and the results are shown in Table 7. As shown in thetable not all SuperLEADS have improved activity compared with theoriginal Leads; some showed decreased activity of some type.

TABLE 6 Schema of LEADs position for SuperLEADS generation Series 1   m1= E41H   m1 + m2 = E41H + Y89H Series 2   m1 = E58Q   m1 + m2 = E58Q +F27V Series 3   m1 = R125H   m1 + m2 = R125H + M111V Series 4   m1 =E159H   m1 + m2 = E159H + Y89H Series 5   m1 = K121Q   m1 + m2 = K121Q +P109A   m1 + m2 + m3 = K121Q + P109A + K133Q Series 6   m1 = E78H   m1 +m2 = E78H + R33H   m1 + m2 + m3 = E78H + R33H + E58H   m1 + m2 + m3 + m4= E78H + R33H + E58H + L11OV

TABLE 7 SuperLEADs of IFNα-2b multiple mutants SEQ Proteolysis IFNantiviral Mutant ID No. protection activity E41H 88 Pseudo wt IncreasedY89H 1303 Pseudo wt Pseudo wt E41H/Y89H/N45D 979 Increased IncreasedE58Q 89 Increased Pseudo wt F27V 83 Pseudo wt Pseudo wt E58Q/F27V 981Increased Pseudo wt R125H 106 Increased Increased M111V 978 Pseudo wtPseudo wt R125H/M111V 986 Increased Increased E159H 125 Y89H 1303E159H/Y89H 987 K121Q 104 Increased Pseudo wt P109A 97 Pseudo wt Pseudowt K133Q 114 Increased Increased K121Q/P109A 983 Increased Pseudo wtK121Q/P109A/ 984 Increased Increased K133Q/G102R E78H 93 IncreasedIncreased R33H 86 Pseudo wt Pseudo wt E58H 90 Increased Increased L110V98 Pseudo wt Pseudo wt E78H/R33H/ 982 Decreased Decreased E58H/L110VFour mutants with mutations in addition to those selected by therational mutagenesis were generated in the E. coli MutS strain and weredetected by sequencing. The mutants were the following: E41Q/D94G;L117V/A139G; E41H/Y89H/N45D; and K121Q/P109A/K133Q/G102R.

Example 6 Cloning of IFN β in pNAUT, a Mammalian Cell Expression Plasmid

The cDNA encoding IFN β (see, SEQ ID No. 196) was cloned into amammalian expression vector, prior to the generation of the selectedmutations (see, FIGS. 6(O)-6(S) and 8(A). A collected of predesigned,targeted mutants was then generated such that each individual mutant wascreated and processed individually, physically separated form each otherand in addressable arrays. The mammalian expression vector pSSV9 CMV 0.3pA (see, Example 1) was engineered as follows:

The pSSV9 CMV 0.3 pA was cut by PvuII and religated (this step gets ridof the ITR functions), prior to the introduction of a new EcoRIrestriction site by Quickchange mutagenesis (Stratagene). Theoligonucleotide sequences used, follow:

EcoRI forward primer: (SEQ ID NO: 218)5′-GCCTGTATGATTTATTGGATGTTGGAATTCCCTGATGCGGTATTTTC TCCTTACG-3′EcoRI reverse prime: (SEQ ID NO: 219)5′-CGTAAGGAGAAAATACCGCATCAGGGAATTCCAACATCCAATAAATC ATACAGGC-3′

The construct sequence was confirmed by using the followingoligonucleotides:

Seq ClaI forward primer: (SEQ ID NO: 220)5′-CTGATTATCAACCGGGGTACATATGATTGACATGC-3′ Seq XmnI reverse primer(SEQ ID NO: 221) 5′-TACGGGATAATACCGCGCCACATAGCAGAAC-3′.

Then, the XmnI-ClaI fragment containing the newly introduced EcoRI sitewas cloned into pSSV9 CMV 0.3 pA to replace the corresponding wild-typefragment and produce construct pSSV9-2EcoRI.

The IFN β-cDNA was obtained from the pIFNβ1 (ATCC) construct. Thesequence of the IFN β-cDNA was confirmed by sequencing using the primersbelow:

Seq forward primer: 5′-CCTGATGAAGGAGGACTC-3′ (SEQ ID NO:222)Seq reverse primer: 5′-CCAAGCAGCAGATGAGTC-3′. (SEQ ID NO:223)

The verified IFN β-encoding cDNA first was cloned into the pTOPO-TAvector (Invitrogen). After checking of the cDNA sequence by automaticDNA sequencing, the HindIII-XbaI fragment containing the IFN cDNA wassubcloned into the corresponding sites of pSSV9-2EcoRI, leading to theconstruct pAAV-EcoRI-IFNbeta (pNB-AAV-IFN beta) Finally the fragment PvuII of plasmid pNB-AAV-IFN beta was subcloned in PvuII site of pUC 18leading the final construct pUC-CMVIFNbetapA called pNAUT-IFNbeta.

Production and Normalization of IFNβ in Mammalian Cells

IFN β was produced in CHO Chinese Hamster Ovarian cells (obtained fromATCC), using Dulbecco's modified Eagle's medium supplemented withglucose (4.5 g/L; Gibco-BRL) and fetal bovine serum (5%, Hyclone). Cellswere transiently transfected as follows: 0.6×10⁵ cells were seeded into6 well plates and grown for 24 h before transfection. Confluent cells atabout 70%, were supplemented with 1.0 μg of plasmid (from the library ofIFN β mutants) by Lipofectamine Plus reagent (Invitrogen). After gentlyshaking, cells were incubated for 24 h with 1 ml of culture mediumsupplemented with 1% of serum. IFN β was obtained from culturesupernatants 24 h after transfection and stored in aliquots at −80° C.until use.

Preparations of IFN β produced from transfected cells were screenedfollowing sequential biological assays as follows. Normalization of IFNβ concentration from culture supernatants was performed by ELISA. IFN βconcentrations from wild type, and mutant samples were estimated byusing an international reference standard provided by the NIBSC, UK.

Screening and In Vitro Characterization of IFN β Mutants

Two activities were measured directly on IFN samples: antiviral andantiproliferation activities. Dose (concentration)-response (activity)experiments for antiviral or antiproliferation activity allowed for thecalculation of the “potency” for antiviral and antiproliferationactivities, respectively. Antiviral and antiproliferation activitiesalso were measured after incubation with proteolytic samples such asspecific proteases, mixtures of selected proteases, human serum or humanblood. Assessment of activity following incubation with proteolyticsamples allowed to determine the residual (antiviral orantiproliferation) activity and the respective kinetics of half-lifeupon exposure to proteases.

Antiviral Activity—Measured by Cytopathic Effects (CPE)

Antiviral activity of IFN β was determined by the capacity of thecytokine to protect HeLa cells against EMC (mouse encephalomyocarditis)virus-induced cytopathic effects. The day before, HeLa cells (2×10⁵cells/ml) were seeded in flat-bottomed 96-well plates containing 100μl/well of Dulbecco's MEM-GlutamaxI-sodium pyruvate medium supplementedwith 5% SVF and 0.2% of gentamicin. Cells were growth at 37° C. in anatmosphere of 5% CO₂ for 24 hours.

Two-fold serial dilutions of interferon samples were made with MEMcomplete media into 96-Deep-Well plates with final concentration rangingfrom 1600 to 0.6 pg/ml. The medium was aspirated from each well and 100μl of interferon dilutions were added to HeLa cells. Each interferonsample dilution was assessed in triplicate. The two last rows of theplates were filled with 100 μl of medium without interferon dilutionsamples in order to serve as controls for cells with and without virus.

After 24 hours of growth, a 1/1000 EMC virus dilution solution wasplaced in each well, except for the cell control row. Plates werereturned to the CO₂ incubator for 48 hours. Then, the medium wasaspirated and the cells were stained for 1 hour with 100 μl of Bluestaining solution to determine the proportion of intact cells. Plateswere washed in a distilled water bath. The cell bound dye was extractedusing 100 μl of ethylene-glycol mono-ethyl-ether (Sigma). The absorbanceof the dye was measured using an Elisa plate reader (Spectramax). Theantiviral activity of IFN β samples (expressed as number of IU/mg ofproteins) was determined as the concentration needed for 50% protectionof the cells against EMC virus-induced cytopathic effects. Forproteolysis experiments, each point of the kinetic was assessed at 800and 400 pg/ml in triplicate.

Anti-Proliferative Activity

Anti-proliferative activity of IFN β was determined by assessing thecapacity of the cytokine to inhibit proliferation of Daudi cells. Daudicells (1×10⁴ cells) were seeded in flat-bottomed 96-well platescontaining 50 μl/well of RPMI 1640 medium supplemented with 10% SVF, Xglutamine and 1 ml of gentamicin. No cell was added to the last row (“H”row) of the flat-bottomed 96-well plates in order to evaluate backgroundabsorbance of culture medium.

At the same time, two-fold serial dilutions of interferon samples weremade with RPMI 1640 complete media into 96-Deep-Well plates with finalconcentration ranging from 6000 to 2.9 pg/ml. Interferon dilutions (50μl) were added to each well containing 50 μl of Daudi cells. The totalvolume in each well should now be 100 μl. Each interferon sampledilution was assessed in triplicate. Each well of the “G” row of theplates was filled with 50 μl of RPMI 1640 complete media in order to beused as positive control. The plates were incubated for 72 hours at 37°C. in a humidified, 5% CO₂ atmosphere.

After 72 hours of growth, 20 μl of Cell titer 96 Aqueous one solutionreagent (Promega) was added to each well and incubated 1H30 at 37° C. inan atmosphere of 5% CO₂. To measure the amount of colored solubleformazan produced by cellular reduction of the MTS, the absorbance ofthe dye was measured using an Elisa plate reader (Spectramax) at 490 nmn.

The corrected absorbances (“H” row background value subtracted) obtainedat 490 nm were plotted versus concentration of cytokine. The ED50 valuewas calculated by determining the X-axis value corresponding to one-halfthe difference between the maximum and minimum absorbance values.(ED50=the concentration of cytokine necessary to give one-half themaximum response).

Treatment of IFN β with Proteolytic Preparations

Mutants were treated with proteases in order to identify resistantmolecules. The resistance of the mutant IFN β molecules compared towild-type IFN β against enzymatic cleavage (120 min, 25° C.) by amixture of proteases (containing 1.5 pg of each of the followingproteases (1% wt/wt, Sigma): α-chymotrypsin, carboxypeptidase,endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C,endoproteinase Lys-C, and trypsin) was determined. At the end of theincubation time, 10 μl of anti-proteases complete, mini EDTA free, Roche(one tablet was dissolved in 10 ml of DMEM and then diluted to 1/1000)was added to each reaction in order to inhibit protease activity.Treated samples were then used to determine residual antiviral orantiproliferation activities.

Protease Resistance—Kinetic Analysis

The percent of residual IFN β activity over time of exposure toproteases was evaluated by a kinetic study using 1.5 pg of proteasemixture. Incubation times were: 0 h, 0.5 h, 2 h, 4 h, 8 h, 12 h, 24 hand 48 h. Briefly, 20 μl of each proteolytic sample (proteases, serum,blood) was added to 100 μl of IFN β at 400 and 800 pg/ml and incubatedfor variable times, as indicated. At the appropriate time points, 10 μlof anti-proteases mixture, mini EDTA free, Roche (one tablet wasdissolved in 10 ml of DMEM and then diluted to 1/500) was added to eachwell in order to stop proteolysis reactions. Biological activity assayswere then performed as described for each sample in order to determinethe residual activity at each time point.

Performance

The various biological activities, protease resistance and potency ofeach individual mutant were analyzed using a mathematical model andalgorithm (NautScan™; Fr. Patent No. 9915884; see, also publishedInternational PCT application No. WO 01/44809 based on PCT noPCT/FR00/03503). Data was processed using a Hill equation-based modelthat uses key feature indicators of the performance of each individualmutant. Mutants were ranked based on the values of their individualperformance and those on the top of the ranking list were selected asleads.

Using the 2D-scanning and 3D-scanning methods described above inaddition to the 3-dimensional structure of IFNβ, the following aminoacid target positions were identified as is-HITs on IFNβ, whichnumbering is that of the mature protein (SEQ ID NO:196):

By 3D-scanning (see, SEQ ID Nos. 234-289, 989-1015): D by Q at position39, D by H at position 39, D by G at position 39, E by Q at position 42,E by H at position 42, K by Q at position 45, K by T at position 45, Kby S at position 45, K by H at position 45, L by V at position 47, L byI at position 47, L by T at position 47, L by Q at position 47, L by Hat position 47, L by A at position 47, K by Q at position 52, K by T atposition 52, K by S at position 52, K by H at position 52, F by I atposition 67, F by V at position 67, R by H at position 71, R by Q atposition 71, D by H at position 73, D by G at position 73, D by Q atposition 73, E by Q at position 81, E by H at position 81, E by Q atposition 107, E by H at position 107, K by Q at position 108, K by T atposition 108, K by S at position 108, K by H at position 108, E by Q atposition 109, E by H at position 109, D by Q at position 110, D by H atposition 110, D by G at position 110, F by I at position 111, F by V atposition 111, R by H at position 113, R by Q at position 113, L by V atposition 116, L by I at position 116, L by T at position 116, L by Q atposition 116, L by H at position 116, L by A at position 116, L by V atposition 120, L by I at position 120, L by T at position 120, L by Q atposition 120, L by H at position 120, L by A at position 120, K by Q atposition 123, K by T at position 123, K by S at position 123, K by H atposition 123, R by H at position 124, R by Q at position 124, R by H atposition 128, R by Q at position 128, L by V at position 130, L by I atposition 130, L by T at position 130, L by Q at position 130, L by H atposition 130, L by A at position 130, K by Q at position 134, K by T atposition 134, K by S at position 134, K by H at position 134, K by Q atposition 136, K by T at position 136, K by S at position 136, K by H atposition 136, E by Q at position 137, E by H at position 137, Y by H atposition 163, Y by I at position 163, R by H at position 165, R by Q atposition 165.

By 2D-scanning (see, SEQ ID Nos.1016-1302, and table above): M by V atposition 1, M by I at position 1, M by T at position 1, M by Q atposition 1, M by A at position 1, L by V at position 5, L by I atposition 5, L by T at position 5, L by Q at position 5, L by H atposition 5, L by A at position 5, F by I at position 8, F by V atposition 8, L by V at position 9, L by I at position 9, L by T atposition 9, L by Q at position 9, L by H at position 9, L by A atposition 9, R by H at position 11, R by Q at position 11, F by I atposition 15, F by V at position 15, K by Q at position 19, K by T atposition 19, K by S at position 19, K by H at position 19, W by S atposition 22, W by H at position 22, N by H at position 25, N by S atposition 25, N by Q at position 25, R by H position 27, R by Q position27, L by V at position 28, L by I at position 28, L by T at position 28,L by Q at position 28, L by H at position 28, L by A at position 28, Eby Q at position 29, E by H at position 29, Y by H at position 30, Y byI at position 30, L by V at position 32, L by I at position 32, L by Tat position 32, L by Q at position 32, L by H at position 32, L by A atposition 32, K by Q at position 33, K by T at position 33, K by S atposition 33, K by H at position 33, R by H at position 35, R by Q atposition 35, M by V at position 36, M by I at position 36, M by T atposition 36, M by Q at position 36, M by A at position 36, D by Q atposition 39, D by H at position 39, D by G at position 39, E by Q atposition 42, E by H at position 42, K by Q at position 45, K by T atposition 45, K by S at position 45, K by H at position 45, L by V atposition 47, L by I at position 47, L by T at position 47, L by, Q atposition 47, L by H at position 47, L by A at position 47, K by Q atposition 52, K by T at position 52, K by S at position 52, K by H atposition 52, F by I at position 67, F by V at position 67, R by H atposition 71, R by Q at position 71, D by Q at position 73, D by H atposition 73, D by G at position 73, E by Q at position 81, E by H atposition 81, E by Q at position 85, E by H at position 85, Y by H atposition 92, Y by I at position 92, K by Q at position 99, K by T atposition 99, K by S at position 99, K by H at position 99, E by Q atposition 103, E by H at position 103, E by Q at position 104, E by H atposition 104, K by Q at position 105, K by T at position 105, K by S atposition 105, K by H at position 105, E by Q at position 107, E by H atposition 107, K by Q at position 108, K by T at position 108, K by S atposition 108, K by H at position 108, E by Q at position 109, E by H atposition 109, D by Q at position 110, D by H at position 110, D by G atposition 110, F by I at position 111, F by V at position 111, R by H atposition 113, R by Q at position 113, L by V at position 116, L by I atposition 116, L by T at position 116, L by Q at position 116, L by H atposition 116, L by A at position 116, L by V at position 120, L by I atposition 120, L by T at position 120, L by Q at position 120, L by H atposition 120, L by A at position 120, K by Q at position 123, K by T atposition 123, K by S at position 123, K by H at position 123, R by H atposition 124, R by Q at position 124, R by H at position 128, R by Q atposition 128, L by V at position 130, L by I at position 130, L by T atposition 130, L by Q at position 130, L by H at position 130, L by A atposition 130, K by Q at position 134, K by T at position 134, K by S atposition 134, K by H at position 134, K by Q at position 136, K by T atposition 136, K by S at position 136, K by H at position 136, E by Q atposition 137, E by H at position 137, Y by H at position 138, Y by I atposition 138, R by H at position 152, R by Q at position 152, Y by H atposition 155, Y by I at position 155, R by H at position 159, R by Q atposition 159, Y by H at position 163, Y by I at position 163, R by H atposition 165, R by Q at position 165, M by D at position 1, M by E atposition 1, M by K at position 1, M by N at position 1, M by R atposition 1, M by S at position 1, L by D at position 5, L by E atposition 5, L by K at position 5, L by N at position 5, L by R atposition 5, L by S at position 5, L by D at position 6, L by E atposition 6, L by K at position 6, L by N at position 6, L by R atposition 6, L by S at position 6, L by Q at position 6, L by T atposition 6, F by E at position 8, F by K at position 8, F by R atposition 8, F by D at position 8, L by D at position 9, L by E atposition 9, L by K at position 9, L by N at position 9, L by R atposition 9, L by S at position 9, Q by D at position 10, Q by E atposition 10, Q by K at position 10, Q by N at position 10, Q by R atposition 10, Q by S at position 10, Q by T at position 10, S by D atposition 12, S by E at position 12, S by K at position 12, S by R atposition 12, S by D at position 13, S by E at position 13, S by K atposition 13, S by R at position 13, S by N at position 13, S by Q atposition 13, S by T at position 13, N by D at position 14, N by E atposition 14, N by K at position 14, N by Q at position 14, N by R atposition 14, N by S at position 14, N by T at position 14, F by D atposition 15, F by E at position 15, F by K at position 15, F by R atposition 15, Q by D at position 16, Q by E at position 16, Q by K atposition 16, Q by N at position 16, Q by R at position 16, Q by S atposition 16, Q by T at position 16, C by D at position 17, C by E atposition 17, C by K at position 17, C by N at position 17, C by Q atposition 17, C by R at position 17, C by S at position 17, C by T atposition 17, L by N at position 20, L by Q at position 20, L by R atposition 20, L by S at position 20, L by T at position 20, L by D atposition 20, L by E at position 20, L by K at position 20, W by D atposition 22, W by E at position 22, W by K at position 22, W by R atposition 22, Q by D at position 23, Q by E at position 23, Q by K atposition 23, Q by R at position 23, L by D at position 24, L by E atposition 24, L by K at position 24, L by R at position 24, W by D atposition 79, W by E at position 79, W by K at position 79, W by R atposition 79, N by D at position 80, N by E at position 80, N by K atposition 80, N by R at position 80, T by D at position 82, T by E atposition 82, T by K at position 82, T by R at position 82, I by D atposition 83, I by E at position 83, I by K at position 83, I by R atposition 83, I by N at position 83, I by Q at position 83, I by S atposition 83, I by T at position 83, N by D at position 86, N by E atposition 86, N by K at position 86, N by R at position 86, N by Q atposition 86, N by S at position 86, N by T at position 86, L by D atposition 87, L by E at position 87, L by K at position 87, L by R atposition 87, L by N at position 87, L by Q at position 87, L by S atposition 87, L by T at position 87, A by D at position 89, A by E atposition 89, A by K at position 89, A by R at position 89, N by D atposition 90, N by E at position 90, N by K at position 90, N by Q atposition 90, N by R at position 90, N by S at position 90, N by T atposition 90, V by D at position 91, V by E at position 91, V by K atposition 91, V by N at position 91, V by Q at position 91, V by R atposition 91, V by S at position 91, V by T at position 91, Q by D atposition 94, Q by E at position 94, Q by Q at position 94, Q by N atposition 94, Q by R at position 94, Q by S at position 94, Q by T atposition 94, I by D at position 95, I by E at position 95, I by K atposition 95, I by N at position 95, I by Q at position 95, I by R atposition 95, I by S at position 95, I by T at position 95, H by D atposition 97, H by E at position 97, H by K at position 97, H by N atposition 97, H by Q at position 97, H by R at position 97, H by S atposition 97, H by T at position 97, L by D at position 98, L by E atposition 98, L by K at position 98, L by N at position 98, L by Q atposition 98, L by R at position 98, L by S at position 98, L by T atposition 98, V by D at position 101, V by E at position 101, V by K atposition 101, V by N at position 101, V by Q at position 101, V by R atposition 101, V by S at position 101, V by T at position 101, M by C atposition 1, L by C at position 6, Q by C at position 10, S by C atposition 13, Q by C at position 16, L by C at position 17, V by C atposition 101, L by C at position 98, H by C at position 97, Q by C atposition 94, V by C at position 91, N by C at position 90.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A modified interferon beta (IFN beta) cytokine, wherein said modifiedIFN beta cytokine differs from an unmodified IFN beta cytokine by twoamino acid substitutions, wherein said unmodified IFN beta cytokinecomprises the amino acid sequence of SEQ ID NO: 196, said two amino acidsubstitutions being either: (a) substitution of the 5th and 47thpositions in SEQ ID NO: 196, with aspartic acid and isoleucine,respectively; or (b) substitution of the 5th and 47th positions in SEQID NO: 196, with asparagine and isoleucine, respectively; or (c)substitution of the 6th and 47th positions in SEQ ID NO: 196, withglutamine and isoleucine, respectively, and wherein said two amino acidsubstitutions confer increased resistance to proteolysis over theunmodified IFN beta cytokine.
 2. The modified IFN beta cytokine of claim1, wherein only the primary amino acid sequence of said unmodified IFNbeta cytokine is modified.
 3. A pharmaceutical composition comprisingthe modified IFN beta cytokine of claim
 1. 4. The pharmaceuticalcomposition of claim 3, further comprising a pharmaceutically acceptablecarrier or excipient.
 5. The pharmaceutical composition of claim 4,wherein the pharmaceutically acceptable carrier or excipient is selectedfrom the group consisting of a binding agent, a filler, a lubricant, adisintegrant, and a wetting agent.
 6. The pharmaceutical composition ofclaim 3, further comprising a pharmaceutically acceptable additive. 7.The pharmaceutical composition of claim 6, wherein the pharmaceuticallyacceptable additive is selected from the group consisting of asuspending agent, an emulsifying agent, a non-aqueous vehicle, and apreservative.
 8. The pharmaceutical composition of claim 3, wherein thecomposition is in the form of a liquid, a solution, a suspension, anaerosol, a tablet, a lozenge or a capsule.
 9. The pharmaceuticalcomposition of claim 8, wherein the composition is in the form of atablet or a capsule.
 10. The pharmaceutical composition of claim 3,wherein said composition is formulated for oral, parenteral,intravenous, intradermal, subcutaneous, buccal, inhalation,intramuscular, rectal or topical administration.
 11. The pharmaceuticalcomposition of claim 10, wherein said composition is formulated for oraladministration.
 12. The pharmaceutical composition of claim 11, whereinthe pharmaceutical composition is formulated for oral administration tothe mouth or gastrointestinal tract.
 13. The pharmaceutical compositionof claim 3, wherein the pharmaceutical composition is formulated forcontrolled-release of the modified IFN beta cytokine.